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are capable of dissimilating nitrates and forming nitro- gen oxides. Research into fungal nitrate reduction under conditions of hypoxia and anoxia is an important.
ISSN 0003-6838, Applied Biochemistry and Microbiology, 2007, Vol. 43, No. 5, pp. 544–549. © Pleiades Publishing, Inc., 2007. Original Russian Text © E.V. Morozkina, A.V. Kurakov, 2007, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 5, pp. 607–613.

Dissimilatory Nitrate Reduction in Fungi under Conditions of Hypoxia and Anoxia: A Review E. V. Morozkinaa and A. V. Kurakovb a

Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071 Russia; e-mail: [email protected] b Moscow State University, Moscow, 119992 Russia; e-mail: [email protected] Received January 15, 2007

Abstract—Recent progress in studies of anaerobic nitrate reduction and nitrous oxide formation in fungi has been reviewed. Current understanding of the biochemistry of nitrate and nitrite reduction to nitrous oxide and ammonium under oxygen limitation is presented, with emphasis on patterns of fungal co-denitrification, properties of the enzymes involved, and prevalence of nitrate respiration among fungal species. DOI: 10.1134/S0003683807050079

The capacity for nitrate respiration is widespread among bacteria. Under oxygen deficiency in the environment, denitrifying bacteria derive energy from electron transport phosphorylation coupled to reduction of – – nitrogen oxides (N O 3 , N O 2 , NO, and N2O). Species have been identified, possessing a complete set of reductases, which, nevertheless, reduce nitrogen to nitrites and nitrous oxide via truncated chains. Nitrate, nitrite, NO-, and N2O-reductases are sequentially involved into the process, comprising the complete chain of nitrogen respiration (i.e., dissimilatory reduction of nitrates to gaseous derivatives of nitrogen, such as NO, N2, and N2O) [1]. In certain bacteria, dissimilatory reduction of nitrates to ammonium are catalyzed by nitrate and nitrite reductases. In 1990s, it was established that, in addition to bacteria, certain eukaryotic organisms, particularly fungi, are capable of dissimilating nitrates and forming nitrogen oxides. Research into fungal nitrate reduction under conditions of hypoxia and anoxia is an important problem of contemporary physiology and biochemistry. In this paper, we sought to review recent findings on nitrate reduction and nitrous oxygen formation in fungi under conditions of decreased partial pressure of oxygen and discuss current understanding of the underlying biochemical processes. DENITRIFICATION OF NITRATES/NITRITES TO NITROUS OXIDE IN FUNGI Until the 1990s, data on the formation of nitrous oxide (N2O) by fungi were limited to several reports [2–4]. Although the reported conditions that favored the release of N2O by fungal cultures were dissimilar, it would seem most convincing that its formation was associated with nitrate or nitrite reduction under anaer-

obic or microaerobic conditions. This assumption was corroborated by identification of a nitrate-/nitriteinduced and aeration-repressed cytochrome P-450 (which was temporary termed P-450dNIR) in the fungi Fusarium oxysporum and Cylindrocarpon tonkinense, which both form nitrous oxide [5, 6]. Subsequent studies demonstrated that many species of micromycetes (belonging to the classes Ascomycetes and Deuteromycetes) are capable of forming nitrous oxide, when grown on nitrite-containing media under anaerobic conditions [6, 7]. This capacity was observed less frequently, if the fungi were grown on nitrate-containing media [8]. The presence of ammonium in the medium did not inhibit the process of nitrogenous oxide release [8]. As demonstrated in [9], introduction of ammonium into the medium increased the formation of N2O from nitrates 1.5- to 2.5-fold. Shoun et al. found that aeration suppresses the nitrate-/nitrite-induced cytochrome P-450nor, which functions as a fungal NO reductase. This effect was not observed on addition of ammonium [5, 10]. Acetylene, a nitrous oxide reductase in denitrifying bacteria, failed to affect N2O formation in fungi [7, 11]. These observations indicate that N2O is the major final product of anaerobic reduction of nitrites and nitrates inn micromycetes. The fungi of the genera Fusarium, Chaetonium, Humicola, Cylindrocarpon, Trichoderma, etc. (typical inhabitants of soil and rhizosphere, which degrade plant debris) are characterized by a relatively pronounced ability to form N2é. Under optimum conditions, the contribution of fungi to nitrous oxide release from soils was in the range 1–8%, which indicates that fungal denitrification holds important implications for ecology [12, 13]. Experiments with acidic brown soils into which nitrogenous fertilizers were introduced demonstrated that fungi are involved in N2O production to a considerably greater extent [14].

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NO reductase. NO reductase is the enzyme that catalyzes the formation of N2O from NO in the course of denitrification. Data from the literature indicate that, in fungi, these processes are underlain by P-450nor, which induced by nitrate and nitrite. The activity of NO reductase isolated from the fungi Fusarium oxysporum and Cylindrocarpon tonkinense amounted to 300 µmol/min per 1 mg protein, which is more than five times higher than the activity of bacterial NO reductases [1, 5, 6, 10, 15]. The mechanism of action of the enzyme is distinct from that of the bacterial NO reductase. P-450nor accepts electrons directly from NADH without the involvement of the electron-transporting chain (contrary to what is observed in bacterial nitrate respiration). NO reductase performs the function of electron transportation, as well as ensures organism detoxification by eliminating NO [5, 16]. In addition, filamentous fungi (including F. oxysporum) synthesize flavohemo– globin, which catalyzes NO conversion to N O 3 , yet another mechanism whereby excess NO (which is toxic to the cells) is removed [17]. Takaya et al. described a mutant strain, in which loss of the ability to synthesize NO reductase and flavohemoglobin was associated with deranged functions of the respiratory system and retarded growth [18]. Shoun et al. isolated and cloned NO reductaseencoding fungal genes and assigned them to CYP55 subfamily: Fnor (P-450norA and P-450norB in F. oxysporum) [19], Cnor1 and Cnor2 (P-450nor1 and P-450nor2 in C. tonkinense) [10, 20], and Tnor (P-450nor in T. cutaneum [21, 22]. Studies of the expression of the gene CYP55, which encodes NO reductase, demonstrated that it is regulated together with the genes of nitrate assimilation. Nitrite serves as a substrate and inducer of the process simultaneously, regulating both assimilation and dissimilation in fungi [18]. Nitrite reductase. Many fungal species are capable of forming nitrous oxide when grown on nitrite. Attempts to identify the enzyme that catalyzes NO synthesis led researchers to the discovery of nitrite reductase, a homodimer having a molecular weight of 41.8 kDa. In particular, the fungi F. oxysporum [23, 24], C. tonkinense [16], and Fellomyces fuzhouensis [25] were found to possess a dissimilatory nitrite reductase, which cata– lyzed the reduction of N O 2 to NO and was associated with ATP synthesis in mitochondria. An azurin-like protein (15 kDa) synthesized in F. oxysporum acts as an electron donor for nitrite reductase [24]. The activity of the enzyme was reversibly inhibited by chelating agents: for example, addition of EDTA caused a 10% decrease in the enzyme activity. Subsequent incubation with 0.1 mmol CuSO4 at room temperature for 10 min completely restored the activity [23]. The formation of N2O was reproduced in vitro in a reaction mixture containing purified nitrite reductase APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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and NO reductase (cytochrome P-450nor). On addition of P-450nor to a mixture containing nitrite reductase, an immediate stoichiometric conversion of nitrite into nitrous oxide was observed [23]. Kobayashi et al. demonstrated that nitrite reductase of F. oxysporum is a soluble enzyme located between the outer and inner membranes of mitochondria [23]. The capacity for co-denitrification—the formation of nitrous oxide and molecular nitrogen from nitrites and other nitrogenous compounds (azide, salicylhydroxamic acid, and ammonium)—is one of the most important properties of fungal dissimilatory nitrite reductases (such as that of F. oxysporum) [26]. During the growth of F. oxysporum on a nitrite-containing medium, addition of azide or (and) salicylic acid increased the rate of nitrogenous oxide formation after a period (la) of adaptation of the fungus to the added compounds; in addition, release of molecular nitrogen was also observed under such conditions. It would seem that the observed increase in N2O formation was related to assimilation of nitrogen from these compounds. The synthesis of the dissimilatory nitrite reductase in fungal cells is induced by nitrates and nitrites, but the enzyme also catalyzes the transfer of nitrosyl groups from nitrite to nucleophilic compounds, e.g., azide. The phenomenon of co-denitrification and the role played by the dissimilatory nitrite reductase in this process have long been known to be characteristic of bacteria [27, 28]. Nitrogen dioxide is the end product of co-denitrification in bacteria, whereas fungi form nitrous oxide and molecular nitrogen [26]. Experiments with labeled compounds [15N]azide, [14N]nitrite, and [14N]salicylic acid demonstrated that both nitrogen atoms in molecular nitrogen (15N2) originate in azide, whereas N2O appears as a mixture of 14N14NO and 15N14NO; in other words every third nitrogen atom in nitrous oxide originates in azide, and at least one comes from nitrite. This, the fungus F. oxysporum may use distinct nitrogen compounds as substrates. Nitrate reductase. Studies of Aspergillus nidulans cultured under conditions of decreased oxygen supply led Bull et al. to suggest that a dissimilatory nitrate reductase may be synthesized (they were the first to advance this hypothesis) [29]. Kurakov et al. observed a considerable increase (of 10 to 14 times) in the activity of nitrate reductase, when the mycelium was grown under anaerobiosis [8, 11, 30] and demonstrated that the increase in the level of nitrate reductase activity depends on the ability of the mycelium to form nitrous oxide in a nitrate-containing medium. Of interest, a sharp increase in the enzyme activity, observed in F. oxysporum 11dn1, grown under anaerobic conditions, occurred irrespective of the form in which the – source of nitrogen was present in the medium (N O 3 , –

+

N O 2 , or N H 4 ), and even in media devoid of nitrogen. The activity of nitrate reductase of the fungus F. oxysporum 11dn1 in a nitrate-containing medium was as

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MOROZKINA, KURAKOV NO–3

CH3CH2OH +

NAD

NADH

NADH

NAD+

Ald

CH3CHO NAD+

NADH

NADH

NAD+

AddA

Nar

NO2– Nir

NH+4

CH3CO–CoA ADP, PI Ack

ATP, CoA

CH3COOH Fig. 1. Tentative mechanism of dissimilatory nitrate reduction to ammonium in fungi [18]: Ald, alcohol dehydrogenase; Add, acetaldehyde dehydrogenase; Ack, acetate kinase; Nar, nitrate reductase; Nir, nitrite reductase; Pi, inorganic phosphate.

high as 0.3–1.8 µmol N O 2 per 1 min per 1 mg protein, which is comparable to that of bacterial dissimilatory nitrate reductases and an order of magnitude higher than that of assimilatory enzymes [11]. –

A membrane-bound reductase isolated from the mitochondrial fraction of the fungus F. oxysporum MT 811 was capable of using ubiquinone as an electron donor (a physiological donor of respiratory chain electrons) [25]. Studies of nitrate reductases of F. oxysporum strains 11dn1 and MT 811, involving determination of the structure of the active centers, molecular weight measurements, and assessment of spectral parameters, revealed considerable similarity of the enzymes to their counterparts (dissimilatory nitrate reductases) from E. coli and other denitrifying bacteria that contain molybdenum cofactor cytochrome b, and FeS clusters [1, 8, 25, 30]. Takaya et al. demonstrated that the fungal NADdependent formate dehydrogenase, unlike its classic counterpart from eukaryotes, as associated with the respiratory chain and transports electrons from formate to nitrate reductase via ubiquinone pool (Fig. 1). The association with formate dehydrogenase demonstrates that the nitrate reductase system of fungi are similar to those of E. coli. ANAEROBIC REDUCTION OF NITRATES TO AMMONIUM In addition to dissimilatory nitrate reduction with the formation of nitrous oxide, the fungi F. oxysporum and A. nidulans exhibit the capacity for dissimilatory anaerobic reduction of nitrates to ammonium [32, 33]. – This process, involving sequential reduction of N O 3 to +

N H 4 , catalyzed by a nitrate/nitrite reductase, occurs in parallel with catabolic oxidation of ethanol (the donor of electrons) to acetate and substrate phosphorylation,

which both support the fungal growth under anaerobic conditions (Fig. 1). The main stages of the process are (1) acetaldehyde dehydrogenase (AddA)-catalyzed conversion of acetaldehyde to acetyl-CoA and (2) subsequent hydrolysis of the latter by the ATP-forming acetate kinase (AcK) [32]. Such reactions, known to occur in obligate anaerobes (bacteria of the genus Clostridium) [34], do not seem to be typical of eukaryotic cells. Nitrate and nitrite reductases involved in the reduction of nitrogen oxides to ammonium use NADH as an electron donor and are assimilatory enzymes. Fifteen out of the 17 tested fungal strains (Talaromyces rotundus, Trichophyton rubrum, Penicillium abeanum, and several others, including the related Fusarium oxysporum) exhibited the capacity for dissimilatory nitrate reduction with the formation of ammonium [32]. With the exception of Hyalodendron sp. (Mastigomycotina) and Podospora carbonaria (Zygomycotina), the fungi were largely represented by ascomycetes or ascomycete-type mitotic species. COMPARISON OF NITRATE DISSIMILATION IN FUNGI AND BACTERIA Under anaerobic conditions (0 µmol é2 per 1 h per 1 g dry biomass), when denitrification takes place in bacteria, fungi reduce nitrates to nitrites and ammonium [9, 35]. It seems that fungi still need some oxygen for inducing denitrification and forming nitrous oxide, whereas its excess suppresses the process. At present, no direct evidence in support of this hypothesis has been obtained. Kobayashi et al. suggested that oxygen is required for oxidative reactions of biosyntheses of sterols, hemes, and polyunsaturated fatty acids. A gain in the biomass of F. oxysporum and C. tonkinense was observed if ergosterol was added into the media under anaerobic denitrifying conditions [16]. Nevertheless, several lines of evidence indicate that, in F. oxysporum, denitrification and oxygenous respiration use the respiratory chain of mitochondria and occur simultaneously under the conditions of limited aeration. For example, nitrate reductase activity coupled to ATP synthesis was found in the mitochondrial fraction of denitrifying cells of F. oxysporum, if malate and pyruvate (or succinate, or formate) are used as electron donors [16]. Activity studies and measurement of specific absorption spectra demonstrated that the mitochondrial fraction contained cytochrome c oxidase. Data on the suppression of fungal denitrification by respiratory inhibitors are also indicative of coupling of processes of denitrification and oxygenous respiration. Out of the four terminal dehydrogenases (nitrate reductase, nitrite reductase, NO reductase, and N2O reductase) comprising the system of denitrification in bacteria, only nitrate reductase is located at the inner side of the plasma membrane and participates in the formation of the transmembrane potential (Fig. 2) [1].

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Unlike the other three reductases located at the periplasmic side of the membrane, this enzyme receives electrons from reduced ubiquinone. Nitrite reductase NO reductase, and N2O reductase receive electrons from cytochrome c, absorbing protons in the periplasm and decreasing the proton-deriving force. These reductases operate in a counterflow to complex III (cytochrome bc1), which generates more proton-driving force than any of the above reductases can absorb. Therefore, in bacteria, electrons are transferred from – – reduced ubiquinone to N O 3 , N O 2 , NO, and N2O via four terminal reductases (nitrate reductase, nitrite reductase, NO reductase, and N2O reductase) with the same stoichiometry, two protons per two electrons [1]. With the exception of nitrite reductase, which absorbs protons in the mitochondrial intermembrane space, the enzymes involved in nitrate reduction in fungal mitochondria have a topology that is similar to that of their bacterial counterparts (Fig. 2). Considering the mixed system of respiration in fungi, electrons leaving complex III may follow two pathways of transport: (1) – to O2 via cytochrome oxidase and (2) to N O 2 via nitrite reductase (Fig. 3). It would seem that the system is constructed in such a way so as to avoid energetically unfavorable reactions after complex III (bacterial type involving NO and N2O reductases) and, for this reason, channels electrons to é2. Fungal nitrate reductase may receive electrons from three sources: (1) formate dehydrogenase, (2) complex I (NADH-dependent ubiquinone reductase), and (3) complex II (succinate dehydrogenase). However, as demonstrated by Japanese researchers, the low activities of complexes I and II indicate that, under the conditions of fungal nitrate respiration, the interaction between formate reductase and nitrate reductase plays a dominant role [25];this means that NADH is accumulated for NO reductase (P-450 reductase), which catalyzes the reaction of detoxification and electron elimination in hypoxia [15]. Thus, the hybrid system of respiration in fungi combines the energetically advantageous nitrate respiration (effected by the pair formate reductase–nitrate reductase) and classic oxygenous respiration (with cytochrome oxidase as the terminal component). Protondriving force is generated in the course of reactions catalyzed by formate dehydrogenase and nitrate reductase. The formation of formate from pyruvate is catalyzed by pyruvate formate-lyase [36]. In fungi, electrons leaving complex III are transported to é2, which results in a higher transmembrane potential than that generated via the classic pathway of nitrate dissimilation in bacteria. Excess electrons formed under the conditions of hypoxia are “shunted” to the molecule of NO, which is then reduced to N2é by NO reductase. Mitochondria were long viewed as cell organelles characteristic of aerobic respiration in eukaryotic organisms. However, identification in mitochondrial of enzymes required for anaerobic respiration calls this APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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Bacteria NO2–

Periplasm NO–3

NO–3

Nar

Nir

NO

Nor

N2O

N2

Cytoplasm

NO2–

Fungi

H2O Mitochondrion NO2–

Nor Nir

NO

NO–3

NO–3

N2O

NO NOD

Na

NO–3

N2Or

NO–3

Cytoplasm

Fig. 2. Comparison of denitrification systems operating in bacteria and fungi [18]: Nar, nitrate reductase; Nir, nitrite reductase; Nor, NO reductase; N2Or, N2O reductase; NOD NO dioxigenase.

statement into question. The problem of the evolutionary relation between fungal and bacterial systems of denitrification remains to be clarified. The location of nitrite reductase if the fungus F. oxysporum in the intermembrane space of mitochondria is similar to the periplasmic arrangement of the majority of bacterial enzymes [16]. According to the theory of John and Whatley, bacterial cells evolved into precursors of promitochondria, which lost certain mechanisms of adaptation (particularly the capacity for denitrification) in the course of evolution [37]. The ability of fungi to dissimilate nitrates and nitrites under anaerobic conditions and localization of key enzymes of denitrification in mitochondria are indicative of the possible bacterial origin of these organelles, providing indirect evidence in support of the above hypothesis. On the other hand, the presence of a unique NO reductase in fungi, which differs considerably from its bacterial counterpart in both the structure and the function, calls the hypothesis into question. Thus, the issue of whether the fungal system of denitrification has a promitochondrial origin or results from recent evolutionary adaptation of fungi to changing environmental conditions remains open. *** Most fungi form ATP in the course of aerobic respiration, However, if oxygen supply is insufficient, alternative pathways of dissimilatory nitrate reduction may be used (scheme). Fungi, particularly F. oxysporum, are capable of denitrifying nitrate (to nitrous oxide) or reducing it anaerobically (to ammonium) [5, 6]. Denitrification in fungi occurs at maximum rates within a narrow range of decreased partial pressure of oxygen [9], whereas ammonium fermentation requires anaerobic conditions [32]. The system of denitrification is located in mitochondria, and its first reactions, catalyzed sequentially by nitrate reductase and nitrite

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MOROZKINA, KURAKOV NO2– +

NO +

Intermembrane Nir HCOO–

+

HOCOO–

+ 2H

4H

C549 2H

FDH UQ

0.5 O2

UQH2 UQ

UQH2

I

II

H2O

UQ UQH2

Nar 2H Cytoplasm

2H NO–3 +

4H

NO2– +

2NO + NADH + P-450nor N2O +

Fig. 3. tentative model of mixed respiration in fungi [31]: Nar, nitrate reductase; Nir, nitrite reductase; FDH, formate dehydrogenase; P-450nor, NADH-dependent NO reductase; C549, cytochrome C549; UQ, oxidized ubiquinone; UQH2; reduced ubiquinone; I, ubiquinone cytochrome c reductase; II, cytochrome oxidase.

reductase, are linked to the respiratory chain and ATP synthesis. The fungal NO reductase, unlike its bacterial counterpart, uses NADH as a direct donor of electrons, regenerates NAD+ and detoxifies nitrogen oxide by converting it into nitrous oxide. The capacity for denitrification of nitrites (with the formation of nitrous

oxide) is widespread among diverse fungal taxa. Denitrification of nitrates is less common in fungi. The second pathway of dissimilation of nitrates consists in their reduction to ammonium, coupled to (1) acetate formation from ethanol and (2) substrate phos-

Schematic representation of dissimilatory and assimilatory nitrate reduction in fungi Dissimilatory pathways Denitrification (energy supply, electron shunting)

Co-denitrification

Respiratory nitrate reduction –



Assimilation Reduction of nitrates to ammonium (electron shunting, detoxification, energy supply) Nitrate reduction –

N O3 N O2 (nitrite is released or further reduced)

Assimilatory nitrate reduction –



N O3

N O2



N O3 N O2 (electron shunting, nitrite is excreted or further reduced)

Assimilatory nitrite reduction –

+

N O2 Respiratory nitrite reduction – N O2

NO (NO is released or further reduced)

Respiratory nitrite reduction Ammonifying nitrite reduction – + (with nitrogen incorporation N O2 N H4 from azide, salicylhydroxamic (electron shunting, acid, and ammonium into N2O) detoxification) – – N O2 + N3 N2O + N2

N H4

Ammonium enters metabolic pathways

Ammonium release from the cell Reduction of nitrogen oxide Denitrification associated with – – to nitrous oxide (detoxificaN O 3 /N O 2 N2O tion, electron shunting) NO N2O APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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phorylation. Many fungal species seem to be capable of effecting dissimilatory reduction of nitrates to ammonium; however, data from the literature are not sufficient for clarifying the problem. NADH-dependent nitrate and nitrite reductases are distinct from their dissimilatory mitochondrial counterparts in both the structure and the function [16], but similar to assimilatory reductases of fungi [18]. In order to maintain their vital activity under anoxia and hypoxia, fungi have developed a complex system of regulation, which makes it possible to use both substrate phosphorylation and dissimilatory nitrate reduction, ACKNOWLEDGMENTS This work was supported in part by the Russian Foundation for Basic Research (project nos. 06-0448557 and 06-04-48527). REFERENCES 1. Zumft, W.G., Microbiol. Mol. Biol. Rev., 1997, vol. 61, pp. 533–616. 2. Yoshida, T. and Alexander, M., Soil Sci., 1971, vol. 111, pp. 307–312. 3. Bollag, J.M. and Tung, G., Soil Biol. Biochem., 1972, vol. 4, pp. 271–276. 4. Bleakley, B.H. and Tiedje, J.M., Appl. Environ. Microbiol., 1982, vol. 44, pp. 1342–1348. 5. Shoun, H. and Tanimoto, T., J. Biol. Chem., 1991, vol. 266, pp. 11078–11082. 6. Shoun, H., Kim, D.H., Uchiyama, H., and Sugiyama, J., FEMS Microbiol. Letts., 1992, vol. 94, pp. 277–282. 7. Kurakov, A.V., Pakhnenko, O.A., Kostina, N.V., and Umarov, M.M., Pochvovedenie, 1997, no. 12, pp. 1497– 1503 [Eur. Soil Sci. (Engl. Transl.), no. 12, pp. 1344– 1349]. 8. Kurakov, A.V., Nosikov, A.N., Skrynnikova, E.V., and L’vov, N.P., Curr. Microbiol., 2000, vol. 41, pp. 114– 119. 9. Zhou, Z., Takaya, N., Sakairi, M.A.C., and Shoun, H., Arch. Microbiol., 2001, vol. 175, pp. 19–25. 10. Usuda, K., Toritsuka, N., Matsuo, Y., Kim, D.H., and Shoun, H., Appl. Environ. Microbiol., 1995, vol. 61, no. 3, pp. 883–889. 11. Kurakov, A.V., Nosikov, A.N., Skrynnikova, E.V., and L’vov, N.P., Final Program and Abstracts of 9th Int. Symp. on Microbial Ecology, Amsterdam: Kluwer, 2001, p. 198. 12. Kurakov, A.V., Trudy Vseros. Konf., posvyashchennoi 100-letiyu so dnya rozhdeniya E.N. Mishustina: “Perspektivy razvitiya pochvennoi biologii” (Proc. All-Russia Conf. Devoted to the 100th Anniversary of E.N. Mishustina “Prospects of Development of Soil Biology”), Moscow: Maks Press, 2001, pp. 133–162. 13. Kurakov, A.V., Plant Nutrition for Food Security, Human Health and Environmental Protection, Beijing: Tsinghua Univ. Press, 2005, pp. 1058–1059.

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