HUNGARIAN JOURNAL OF INDUSTRY AND CHEMISTRY Vol. 48(2) pp. 1–4 (2020) hjic.mk.uni-pannon.hu DOI: 10.33927/hjic-2020-20 COMBINATION OF CHEMICAL AND BIOLOGICAL METHODS FOR EFFECTIVE PLANT PROTECTION DÁVID VOZIK 1 AND KATALIN BÉLAFI-BAKÓ*1 1Research Institute on Bioengineering, Membrane Technology and Energetics, University of Pannonia, Egyetem u. 10, Veszprém, 8200, HUNGARY The application of combined biological and chemical techniques to control undesirable processes in plant cultivation is be- coming ever more important to maintain sustainable and environmentally friendly agricultural systems. Entomopathogenic nematodes and bacteria can provide an effective and special technology in the field of plant protection. Keywords: antimicrobial, entomopathogenic nematodes, bacteria 1. Introduction Nowadays, one of the most important challenges world- wide is the sufficient cultivation of edible plants and crops due to the rising trend in population growth and food demand. Moreover, it is important to solve this problem by implementing sustainable agriculture which prefers natural protection and tries to minimize the inter- vention of radical chemicals. It was confirmed recently that some synthetic pesticides are environmental hazards. Since the biological degradation of certain pesticides is slow, their bioaccumulation might cause significant dam- age to ecosystems, soil, natural waters, etc. Therefore, the idea of protecting crops has been extended and a new con- cept, “Integrated Pest Management” (IPM), introduced which involves chemical, biological and biotechnologi- cal methods together with modern cropping, cultivation and breeding technologies [1] in a way which minimizes any risk of environmental damage. To apply this concept in practice requires compre- hensive knowledge of the crops, fitopathogenic microor- ganisms as well as their enemies, and the behaviour of chemicals (e.g. pesticides) that may be used. A field that has hardly been researched are the so-called ento- mopathogenic nematodes and bacteria which have been studied in Hungary for a significant period of time [2]. 2. Entomopathogenic nematodes and bacteria Entomopathogenic nematodes are a group of thread worms that kill certain insects. They enter – in the form of infective juveniles – into the insects, live as parasites *Correspondence: bako@almos.uni-pannon.hu inside them and cause host mortality within 1-2 days. It has turned out, however, that this is not only caused by the nematodes themselves but certain bacteria play an impor- tant role as well. Entomopathogenic bacteria live in symbiosis with en- tomopathogenic nematodes [3,4]. The nematodes provide shelter for the bacteria, an area of the interior part of the intestine of the infective juveniles is transformed into a bacterial chamber where cells of symbiotic bacteria are located. The relationship is highly specific: e.g. the nema- todes of Steinernema usually carry species of Xenorhab- dus bacteria, while Heterorhabditis carry Photorhabdus bacteria. When entering an insect, infective juveniles release the bacteria, which start multiplying rapidly in the haemolymph. It has been proven that although the bacte- ria are primarily responsible for the mortality of the insect host, the nematodes also produce a toxin which is lethal to the insect. These nematode-bacteria complexes can be applied successfully as biological control agents against insect pests in agriculture [5]. Some of these agents are available commercially, as listed in Table 1, where the target insects, habitats and places of usage are presented [6–8]. 3. Antimicrobial compounds Entomopathogenic bacteria contribute not only to the successful activity of entomopathogenic nematodes but can produce special compounds with an antimicrobial ef- fect as well. The main purpose of producing these com- pounds is to protect the colonized cadaver in the soil [9]. The antimicrobial compounds of entomopathogenic bac- teria have been tested and it would seem that these natural agents show a wide range of bioactivities of medical and https://doi.org/10.33927/hjic-2020-20 mailto:bako@almos.uni-pannon.hu 2 VOZIK AND BÉLAFI-BAKÓ Table 1: Examples of the successful application of nematode-bacteria complexes [6–8] Target Habitat Where Japanese beetle Popillia japonica subterranean lawn, turf black vine weevil Otiorhynchus sulcatus subterranean strawberry plants fungus gnats Lycoriella species, Bradysia species subterranean mushroom production diaprepes root weevil Diaprepes abbreviatus epigeal citrus invasive mole cricket Scapteriscus vicinus epigeal lawn, turf codling moth Cydia pomonella cryptic apple, pome fruit Table 2: Plant pathogens tested Pathogen Effect Ref. Phytophthora nicotianae root rot disease of tobacco [13] Erwinia amylovora fire blight disease of several plants that belong to Rosaceae, e.g. apple, pear, etc. [14] Ralstonia solanacearum brown rot disease of potato [15] agricultural interest, e.g. antibiotic, antimycotic and in- secticidal effects [10,11]. For analytical purposes, Fourier Transform Infrared spectrometry (FTIR) was used to identify these compounds [12]. In our laboratories, secondary metabolites of Xenorhabdus budapestiensis (isolated in Hungary) have been investigated [13]. The bacterium was maintained on a Luria Agar (LA) medium and freshly subcultured. The cells were cultured in 1000 mL flasks and incu- bated on a gyrorotary shaker at 25 ◦C . Then the cells were removed by centrifugation and the supernatant extracted. The cell-free filtrate was further purified by chemical methods (adsorption and filtration) to obtain a peptide-rich fraction. This fraction was applied to test for various plant pathogens, as listed in Table 2, including pathogens of tobacco, apple and potato (the latter of which is widespread in Hungary, causing serious damage to inland agriculture). The investigations involved in vitro bioassays, where the antibacterial activity of the biofraction was deter- mined on solid media. In an agar diffusion test, the tested bacterium was mixed with soft agar poured onto LA plates, then a small hole was made in the centre and the biopreparation of the filtrate added [13]. For the so-called overlay test [13], the antimicrobial preparation was incu- bated on the solid Luria Broth Agar (LA) plate then the pathogenic microorganism in soft agar was spread onto the surface. In both cases, an inhibition zone can be iden- tified and determined, if the biopreparation was effective against the pathogenic strain. An example of the agar dif- fusion bioassay is given in Fig. 1. A picture taken from a successful overlay bioassay test is shown in Fig. 2. In our laboratory, in vitro bioassays proved that the bioprepara- tions containing the antimicrobial compounds could be successfully used against these pathogens. Moreover, in planta (in field trials), a bioassay was conducted using infected apple blossom (Erwinia amylovora) which was treated with the biopreparation. It was concluded to be suitable to reduce the symptoms of the infected plants, thus it can be considered as a promis- Figure 1: Antibacterial effect of the preparation in the agar diffusion test. Figure 2: The antagonistic effect of X. budapestensis against E. amylovora EA1 bacteria in an overlay test. Hungarian Journal of Industry and Chemistry EFFECTIVE PLANT PROTECTION 3 Figure 3: Infected apple blossom treated with the bio- preparation. ing biological agent [14, 15]. This successful treatment is illustrated in Fig. 3. Other than plant pathogens, the peptide-rich fraction of Xenorhabdus budapestiensis has recently been tested against species of fungi in vitro [16] in clinical samples. Candida albicans, Candida lusitaniae, Candida krusei, Candida kefyr, Candida tropicalis and Candida glabrata were used in the research by applying the agar diffusion method. The results proved that every Candida species is sensitive to the preparation, thus it would seem that the fraction has a fungicide impact as well. 4. Conclusion In summary, the symbiotic complex of entomopathogenic nematodes and bacteria can be considered as efficient bi- ological agents to control some insect pests, but the sec- ondary metabolites of the bacteria can be effectively used against other pathogenic bacteria and fungi. 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