Administración Sharding

One of the effective plant disease management strategies is based on the control of metabolic relationships in a plant–pathogen system. This can be reached by using compounds that alter or partially block specifi c pathways of biosynthesis in plants or microorganisms, resulting in derangements in the trophic relationships of pathogens with host plants, or in the production of microbial metabolites related to pathogenicity. Such imbalances may not cause the rapid death of pathogens but can lead to a reduction of their vital capacity or/and impair their pathogenic properties. Similar metabolic effects may be caused by plant inhibitors of fungal and bacterial proteases as described above, as well as by the inhibitors of insect and nematode enzymes.

Statins, secondary metabolites of microbial origin belonging to the polyketide group, represent another class of natural compounds that have recently been discovered to modulate metabolic plant–pathogen interactions. The metabolic pathway targeted by statins is sterol biosynthesis.

Sterols are well known to be ubiquitous and important components of outer membrane and intracellular membranes of eukaryotic organisms and to play essential roles in their physiology. These compounds are required for growth, development and reproduction of plant pathogens. A number of organisms including insects, nematodes and oomycetes are not able to syn-thesize sterols or even their precursors. Sterol-dependent phytopathogens obtain free sterols or intermediates from host plants and may be controlled by compounds inducing alterations in content, availability or composition of plant sterols.

Currently, several chemical fungicides are available that inhibit the syn-thesis of ergosterol in fungi. They prevent cellular membrane formation, stop fungal growth and may suppress sporogenesis in established infection agents. However, statins are natural inhibitors of sterol biosynthesis that pre-sumably can cause similar effects. Statins were found to be produced by different microorganisms, predominantly by fi lamentous fungi. They inhibit the enzyme β-hydroxy-β-methylglutaryl-CoA reductase (HMG-CoA reduc-tase) and prevent sterol biosynthesis at the level of conversion of HMG-CoA to mevalonic acid. Statins are widely applied in medicine as drugs against atherosclerogenic diseases because they effectively decrease the blood cho-lesterol level. Thus, the biological compatibility and safety of statins are well proven.

126 L.A. Shcherbakova

Recently, studies were made to investigate the potential of two statins, lovastatin and compactin, in plant protection against diseases (Dzhavakhiya and Petelina, 2008; Ukraintsteva, 2008). In these studies, both statins were obtained by means of microbial synthesis using the ‘superproducer’ strains Aspergillus terreus 45-50 (lovastatin) and Penicillium citrinum 18-12 (compac-tin); these statins were examined in greenhouse and fi eld experiments with plant treatments and in vitro tests on several pathogenic fungi. As statins are insoluble in water, the authors used aqueous solutions of the statin sodium salts that are referred to below as compactin and lovastatin.

The studied statins were found to possess fungicidal activity in vitro against a number of plant pathogenic fungi. The addition of lovastatin at concentrations from 0.001 to 0.1% to agar media inhibited growth of M. grisea, S. nodorum and Coletotrichum atramentarium. Compactin arrested in vitro growth of Cladosporium cucumerinum, S. nodorum and M. grisea at the same concentration range. In addition, 0.001% compactin solution signifi cantly reduced germination of S. nodorum spores while the 0.01% solution com-pletely suppressed spore germination in this fungus. Among tested plant pathogens, the fungus M. grisea, rice blast agent, showed the highest sensitiv-ity to both statins (for lovastatin IC₅₀ was about 0.003%), while S. nodorum was most resistant to lovastatin (IC₅₀ = 0.02%) and C. cucumerinum tended to compactin resistance. Besides a growth-inhibitory effect, the presence of the statins in nutrition media resulted in a discolouration of fungal mycelia in all fungi, especially in M. grisea and C. atramentarium exposed to lovastatin. This observation suggested suppression of fungal melaninogenesis. In all experi-ments, dicolouration-inducing concentrations of the statins were at least tenfold lower than growth-inhibiting concentrations.

The studied statins were revealed to possess disease-preventing proper-ties. They delay and decrease disease development on treated plants when applied simultaneously or prior to inoculation with pathogens. Lovastatin and compactin protected wheat against S. nodorum and tobacco from A. lon-gipes. In addition, lovastatin showed protective activity against M. grisea on rice, whereas compactin was effective against Puccinia graminis on wheat and P. infestans on potato. Both fungicidal and protective effects were strongly dose-rate dependent, but fungitoxic concentrations also made a toxic impact on plants, whereas protection from diseases was provided by far lower con-centrations that were non-phytotoxic. Thus, almost total or 27–36% S. nodo-rum growth inhibition in vitro was observed at 0.1% and 0.01% lovastatin concentrations in nutrition media, respectively. Both doses showed phyto-toxicity on wheat leaves, but only the higher concentration caused retardant effect on growing plants. When 0.1% lovastatin solution was used for wheat seed soaking for 1.5–2 h, reduction of seedling length averaged 50%, and 0.01% concentration caused no inhibitory infl uence on plant growth. A con-siderable reduction of the disease index was found after application of only 0.0005% lovastatin solution on isolated wheat leaves. The applied concentra-tion did not suppress S. nodorum germinaconcentra-tion and in vitro growth but pre-vented disease development with 94 and 72% protection effi cacy at 3 and 7 days after inoculation, respectively. This suggests that a mechanism other

than fungicidal activity contributes to the protective effect of statins ( Dzhavakhiya and Petelina, 2008).

The resistance of fungi to extreme environmental conditions and biotic stresses is largely determined by their ability to produce protective high- molecular-weight pigments. One of the common fungal pigments in cell walls is melanin. Melanin is a coloured polymer produced by many plant pathogenic fungi, including M. grisea, S. nodorum, C. lagenarium and C. atramentarium, through the pentaketide pathway and depends on the availability of acetyl-CoA. Melanin plays a signifi cant role in the infectivity of these fungi. For instance, the ability of the rice pathogen M. grisea to penetrate into tissues of the host plant is directly associated with the presence of melanin in the fungus.

Strains of M. grisea and C. lagenarium defective in melanin formation lose patho-genicity and are incapable of forming mycelial overgrowth in host plants. Rever-tants restoring wild-colour type regain pathogenicity (Dzhavakhiya et al., 1990).

Interestingly, antimelanogenic compounds, aminoalkylphosphinates, a family of phospho-analogues of natural amino acids, suppress biosynthesis of melanin and some toxic metabolites in the polyketide pathway of M. grisea, and are also fungicidal. The aminoalkylphosphinates serve as analogues of alanine, a precursor to pyruvic acid that is required for melanin biosynthesis.

Exposure of fungi to the phospho-analogues of amino acids inactivates pyru-vate dehydrogenase, thus inhibiting synthesis of acetyl-CoA and melanin (Zhukov et al., 2004) as well as afl atoxins (Khomutov, Khurs, Shcherbakova, Mikityuk, Dzavakhiya and Zhemchuzhina; unpublished data). The discov-ery that non-fungicidal lovastatin and compactin concentrations can induce mycelium de-pigmentation and decrease disease severity on plants suggests that their protective effect may be associated with impairing pathogenicity due to an effect on melanin biosynthesis in causative agents. Statins and fun-gal melanin are both polyketides. Thus, the metabolism of the two com-pounds is interrelated, and it is not improbable that statins can negatively mediate a stage of the polyketide pathway involved in the melanization of plant pathogenic fungi.

Although the mode of protective action of statin is required for under-standing and further research, fi rst small-plot fi eld trials suggest they may be of certain interest from a practical point of view. For example, one pre- planting treatment of potato tubers by soaking in 0.1% compactin solution for half an hour resulted in a 1-month delay of late blight (P. infestans) emergence on plants and a slower course of disease. Only extremely high statin concen-trations of 0.5% undesirably infl uenced plant physiological characteristics. By the end of the growing season, an insignifi cant reduction of potato late blight was observed on plants arising from the treated tubers, but these plants produced fewer diseased tubers. There is reason to suppose that lovastatin and compactin possess anti-phytoviral activity (Ukraintseva, 2008).

Further research of the mechanisms responsible for protective activity could help statins take a fi tting place among the biopesticides of tomorrow.

Lovastatin and compactin per se might serve as base molecules for biochem-ical engineering of active analogues, and the approaches used to studying their plant-protecting properties might be implicated in screening of other

128 L.A. Shcherbakova

pathogen-controlling compounds within the statin group and their deriva-tives. Such screening might lead to the detection of new natural polyketides with high protective activity that would not have an adverse effect on plants.

Since the agricultural application of statin-based formulations does not demand as high a level of statin purifi cation as that in the pharmacological industry, using a relatively simple isolation procedure (Dzhavakhiya, 2008) and the ‘superproducer’ strains suggests that statin-based plant- protection technology would be a more economical and ecologically safe strategy than any method using chemical pesticides.

5.4 Conclusion

The disease-preventing effect of natural compounds is a result of a direct or plant-mediated infl uence on targeted pathogens. The compounds can affect phytopathogens directly, interrupting pivotal metabolic pathways and results in the death of the pathogens (biocidal effect). Some natural sub-stances or their analogues specifi cally infl uence pathways related to patho-genicity and toxigenesis. In the case of a plant-mediated mode of action, natural compounds elicit and activate defence responses in plants that result in induced resistance to diseases. In addition, some biogenic compounds infl uencing plants do not induce resistance but enhance tolerance to patho-gens by improving the physiological state of plants. The structural and func-tional diversity of natural compounds as well as their abundance provide a great potential for bringing plant-protection technologies in line with green consumerization.

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