The theoretical background on application of entomopathogenic fungus Beauveria bassiana as a perspective agent for plant biocontrol



The article is devoted to the current issue in the agricultural sector — the use of an alternative method of pest control — the biological method, more precisely the use of entomopathogenic fungi as a promising agent. The author gives a generalized description of the use of entomopathogenic fungi in the protection of plants. The characteristic features of the life cycle and the mechanism of action of the entomopathogenic fungi Beauveria sp. (Beauveria bassiana) as well as its role in Integrated Pest Management (IPM) are distinguished and described. The article analyzes the views of domestic researchers on this topic. In particular, the practical experience of the Kazakh Scientific Research Institute of Plant Protection and Quarantine is generalized.

Key words: biological control, entomopathogenic fungi, Beauveria bassiana, Integrated Pest Management (IPM).

Protection of plants from harmful organisms plays an important role in increasing production and improving the quality of agricultural products. The higher the harvest is expected, the more funds must be spent to obtain it, including protection from pests. At present, plant protection has a complex of methods and means, primarily powerful chemical means for suppressing the number of different groups of pests.

The creation and use of synthetic preparations on a large scale played an outstanding role in the protection of plants, gave a huge economic benefit and led to a significant increase in world food production and raw materials for industry. However, very soon the negative aspects and consequences of the widespread use of chemical plant protection products began to appear:

‒ Accumulation in soil, reservoirs;

‒ The emergence of pesticide-resistant populations of pests;

‒ The emergence of new economically significant pests that formerly existed only as a species (neutral);

‒ Destructive effect on entomophages, pollinators and other kinds of useful fauna;

‒ Threat to human health and livestock,

‒ Violation of natural connections in biocenoses and other phenomena.

As the unilateral use of synthetic pesticides on the biosphere was adversely affected, the problem of finding new ways to control pests, diseases and weeds in addition to traditional methods was becoming more acute. It was obvious that this search should be conducted not only taking into account economic parameters, but also the ecological, sanitary and social aspects of plant protection in general. [1]

Recent years are marked by a more thoughtful and prudent attitude to the chemical method and an increasing interest in biological methods, the desire to shift part of the work on plant protection to nature itself, especially to natural enemies of pests. In this comparative table, the advantages and disadvantages of the two main methods of plant protection are considered (Table 1).

Table 1

Advantages and disadvantages of the two main methods of plant protection

	Chemicalcontrol	Biologicalcontrol
Advantages	• Pests are destroyed quickly and directly on the plants and seeds; • Relatively cheap.	• People are not poisoned by pesticides; • Eco-friendly products are obtained; • High demand in market; • Lack of pest resistance; • Increase of crop yields; • Quality improvement of agricultural products; • Improving soil fertility, improvement of soil microbiota; • The possibility of eliminating the use of chemicals; • Do not have a stressful influence on plants.
Disadvantages	• Toxic to humans and warm-blooded animals; • Environmental pollution; • Leads to appearance of pest resistance; • Accumulation of chemicals in environment and food; • Reduces soil fertility; • Inhibits the growth of plants and the beginning of fruiting; • Decrease of crop yields; • Degrade the quality of agricultural products.	• No immediate effect; • The need for constant monitoring; • The effectiveness is highly dependent on environmental conditions; • Relatively expensive.

Along with this, the combination of agrotechnical, biological, chemical and other methods of plant protection from pests, diseases and weeds in creating differential protective systems allows preserving (at least partially) useful entomophages and reducing the consumption of pesticides. Integrated Pest Management — is the use of all pest control tools with the predominant use of natural control methods.

The use of an integrated protection system allows solving a number of complex problems associated with the use of chemicals, which demonstrates the significant role of the biocontrol:

1. There is no resistance to the means of protection used.
2. There is no «chemical pressing» (phytotoxicity) for plants, the probability of secondary in fections is reduced.
3. Sprayers with integrated protection do not experience stress.
4. Safety for the health of workers in greenhouses.
5. Ecologically pure products that meet the highest requirements.

The main direction of the biological method is the use of their natural enemies — predators, parasites, antagonists — to protect plants from harmful organisms. Major achievements in the field of physiology and biochemistry, ecology and microbiology have led to new promising areas in biological plant protection associated with the use of pheromones, hormones and antibiotics. These changes are reflected in a broader interpretation of the term biological protection of plants, enshrined in the statute of the International Organization of Biological Control, which was adopted in 1971. In this document, a biological method is understood as the use of living organisms or products of their vital activity to prevent or reduce damage caused by harmful organisms. [5]

The use of entomopathogenic fungi in the fight against harmful insects was first discussed at the end of the 19th century. At that time, this prospect became very popular, and for many decades in many countries many attempts were made to reduce the number of harmful insects by artificially spreading pathogenic fungi. Some of these attempts were successful, which proved the possibility of using fungal diseases, but the overwhelming majority of them ended in failure, no doubt because different researchers did not know the conditions necessary for optimal fungal activity.

The literature lists the factors that determine the effectiveness of fungi in the context of biological control. They include the weather conditions, the density of the host population, the conditions in the microbiota of the fungus and host, the stability of the host and virulence of the fungus, the saturation point of the environment by the pathogen, the ease of artificial reproduction and spread of the fungus, the time of application, the ability of the fungus to survive and spread in the insect population, action of fungi on other agents of biological control, the economic importance of such control measures. [2]

One of the most promising groups of entomopathogenic anamorphic ascomycetes, from the point of view of creating preparations for reducing the number of locusts, are the fungi of the genus Beauveria. [7]

As in other entomopathogenic fungi, Beauveria species attack their host insects percutaneously (Fig.1). The infection pathway consists of the following steps: (1) attachment of the spore to the insect cuticle, (2) spore germination on cuticle, (3) penetration through the cuticle, (4) overcoming the host immune response, (5) proliferation within the host, (6) saprophytic outgrowth from the dead host and production of new conidia. [3]

Fig. 1. Infection cycle of B. bassiana

Unlike viruses and many nematodes and bacteria that require specialized routes of entry for infection of insect hosts, entomopathogenic fungi infect via penetration essentially anywhere on the host cuticle, although preferential sites have been noted on various insects. Infection begins with attachment of single-celled dispersive forms of the fungus, e.g., conidia or blastospores, to the insect cuticle. Expression of a variety of hydrolytic enzymes, e.g., proteases, chitinases, and lipases, and other factors, promote germination and growth of the fungus across the surface of the host, and subsequent penetration of cuticular layers. During this process the fungus produces any number of specialized infection structures that can include penetration pegs and/or appressoria, which enable the growing hyphae to penetrate into the host integument. It is essentially in the later stages of this process that the pathogen encounters the host immune system. [4]

B. bassiana produces an array of chemically diverse secondary metabolites, which nominate it as a superior entemopathogenic fungus. Beauvericin, bassianolide, bassianin, tenellin and cyclosporin A are the key secondary metabolites produced by B. bassiana. Investigations on beauvericin have demonstrated that this metabolite has insecticidal, antibiotic, cytotoxic, and ionophoric properties. Bassianin and tenellin are two yellow-colored non-peptide secondary metabolites which inhibit the erythrocyte membrane ATPases. Bassianolide possesses ionophoric and antibiotic activity similar to beauvericin. Bassiacridin is a toxic protein that was purified from a strain of B. bassiana infecting Locusta migratoria at low dosage and causing nearly 50 % mortality. Beauveriolides are potential candidates for treatment of arthroscleroses. [3]

At present, there are no biological preparations based on entomopathogenic fungi in Kazakhstan to control the number of harmful arthropods, and locusts in particular. Kazakh Scientific Research Institute of Plant Protection and Quarantine together with the All-Russian Institute of Plant Protection had the projects on screening new strains of the Beauveria fungus on the basis of virulence on the Moroccan locust, on sucking pests in greenhouses (in manufacturing conditions), assessing the biological activity of strains in the field, combating with the Colorado potato beetle using local strains, determination of the age and phase sensitivity of the Colorado potato beetle to selected strains, and also the efficacy of strains of the entomopathogenic fungus of the genus Beauveria in suppressing the number of larvae of the Colorado potato beetle, etc.

Recently in the Republic of Kazakhstan there has been an expansion of the sector of greenhouse vegetable growing. However, losses of harvest from insect pests in the conditions of greenhouses reach 30–40 %. For example, in most greenhouse farms, the peach aphid Myzodes persicae inflicts perceptible damage on crops. Phytophagous has a high fertility and a short cycle of development. Aphid feeds by sucking out the plant tissue juice, damaged leaves are twisted, deformed, plants are strongly depressed. Chemical protection of plants against aphids is often ineffective due to the rapid adaptation of the pest to insecticides. [6]

Greenhouse whitefly (Trialeurodes vaporariorum) is a broad polyphage, it is able to develop in more than 200 species of plants. Especially great damage to the crop is caused by this pest on cucumbers and tomatoes. Its harmfulness consists not only in the weakening of plants when the larvae are sucking out juices, but also in a sharp decrease in the assimilative capacity of the leaves due to the development of white fungus on the sugary isolates of the sooty fungus. [6]

Ordinary spider mite (Tetranychus urticae) is also a widespread polyphagous pest. The damage caused by the mite is a kind of light dots-nodules. As the number increases and the nutrition of the mites increases, the dots become larger, discolored areas appear and the leaves wither. All this leads to a disruption in the metabolism and even death of plants, and, consequently, to large crop losses. [6]

Therefore, in 2013 in the conditions of the laboratory of biotechnology for testing in greenhouses, small batches (500–1000 g) of four strains of entomopathogenic fungi were developed, showing high activity in relation to spider mite and greenhouse whitefly — 5 kg. In the course of the studies, the biological activity of the produced strains was evaluated for greenhouse whitefly, melon aphid and common spider mite on tomatoes, as well as on roses and cucumbers. The experiments were carried out in the greenhouse farm of KSU named after. Korkyt Ata in the Kyzylorda region. Tests of semi-preparative forms based on strains of the Beauveria bassiana fungus on roses against the greenhouse whitefly were carried out in June 2014. The flow rate of the working fluid was 5 liters per 270 m2. An assessment of the biological efficacy of the selected strain of BCoc2–12 showed that it is most active against the larval stage. The maximum mortality rate of pest larvae was observed on the 7th day after application and was, depending on the titer of the working suspension, from 81.5 to 93.5 %. At the same time, the biological activity of the strain under study did not differ significantly from the model (strain BBK-1) at the same titers of the working suspension. The values of this indicator on adults were significantly lower and did not exceed 64.8 % in all variants of the experiment. Two weeks after treatment, the level of biological activity was significantly lower and varied from 55 to 86.3 %. [8]

The biological activity of the same strain was also assessed for the greenhouse whitefly on tomatoes. The conducted observations showed that the effectiveness of the tested strain, both against larvae and adults, was significantly lower than in the previous experiment. Thus, the decrease in the number of pest larvae in this case on the 7th day after treatment was, depending on the titer, 36.5–79.4 %, and the imago — only 2.1 to 33.5 %. Two weeks after spraying, a similar pattern was observed. Reducing the number of pests at elevated titers of the working suspension was significantly higher than the minimum. In comparison with the model strain, the biological effectiveness of the test culture was significantly lower in almost all cases (except larvae on the 14th day). [8]

The analysis of the obtained data showed significant differences in the biological effectiveness of the fungus depending on its consumption rate. After 14 days, significantly higher biological activity was observed only at the maximum rate of discharge. According to the imagos, only at the maximum concentration one week after treatment the level of population decline was higher compared to the standard. In general, the tests showed that the BCoс2–12 strain, selected at the previous stages of research as the most promising, is able to effectively control the number of greenhouse whitefly during the week at a level of more than 75 % and two weeks — at least 55 %. It should be noted that under the conditions of industrial greenhouses, at elevated temperatures and a lower relative humidity of air, the selected strain of the fungus B. bassiana (BCoс2–12) is able to effectively contain a complex of sucking pests within a week after treatment. [8]

Thus, the entomopathogenic fungus B. bassiana is attracting increased attention as a potential tool for biological control of insect pests. Understanding the mechanisms of fungal pathogenesis in insects will provide a rational basis for strain selection and improvement.

References:

1. Bondarenko N. V. Biologicheskaya zashchita rasteniy [Biological protection of plants]. Vol. 2, Moscow: Agropromizdat, 1986. 278 p.;
2. Emel'yanova N. A., Rukavishnikova B. I. Biologicheskaya bor'ba s vrednymi nasekomymi i sornyakami [Biological control of harmful insects and weeds]. Moscow: Kolos, 1967. 616 p.
3. Chetan Keswani, Surya Pratap Singh and Harikesh Bahadur Singh. Beauveria bassiana: Status, Mode of action, Applications and Safety issues. Biotech Today, Vol. 3, No. 1, pp. 16–19, 2013;
4. Almudena Ortiz-Urquiza and Nemat O. Keyhani. Action on the Surface: Entomopathogenic Fungi versus the Insect Cuticle. Insects, Vol. 4, pp. 357–374, 2013;
5. Shternshis M. V. Biologicheskaya zashchita rasteniy [Biological protection of plants]. Moscow: KolosS, 2004. 264 p.;
6. Informatsionnyy byulleten' MOBB/VPRS. Zashchita rasteniy v usloviyakh zakrytogo grunta: perspektivy ХХI veka [Protection of plants in conditions of a closed ground: prospects of the XXI century. News bulletin of International Organisation for Biological Control — Asia and Pacific Regional Section]. Belarus, 2010. № 41. 223 p.
7. Lednev G. R., Tokarev Yu.S., Uspanov A. M., Kazartsev I. A., Malysh Yu.M., Smagulova, Sh.B., Levchenko M. V., Sagitov A. O. Biologicheskoe raznoobrazie gribov roda Beauveria Rossii i Kazakhstana [Biological diversity of fungi of the genus Beauveria of Russia and Kazakhstan]. Sovremennaya mikologiya v Rossii Vol. 4. 2015. pp. 136–137.
8. Uspanov A. M., Baymagambetov E.Zh., Bolatbekova B.Kh., Lednev G. R. Skrining shtammov entomopatogennykh gribov po priznaku virulentnosti na sosushchikh vreditelyakh zakrytogo grunta v proizvodstvennykh usloviyakh [Screening of strains of entomopathogenic fungi on the basis of virulence on sucking pests of closed ground under production conditions]. Materials of the international scientific conference Innovative ecologically safe technologies of plant protection. Almaty. 2015. pp. 599–604