International Journal of
Molecular Sciences Article
Synthesis of Indoleacetic Acid, Gibberellic Acid and ACC-Deaminase by Mortierella Strains Promote Winter Wheat Seedlings Growth under Different Conditions 1 , Justyna Bohacz 2 , Teresa Korniłłowicz-Kowalska 2 , ´ Ewa Ozimek 1, *, Jolanta Jaroszuk-Sciseł 1 1 Renata Ty´skiewicz , Anna Słomka , Artur Nowak 1 and Agnieszka Hanaka 3 1
2
3
*
Department of Environmental Microbiology, Institute of Microbiology and Biotechnology; Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, 20-033 Lublin, Poland; ´
[email protected] (R.T.);
[email protected] (J.J.-S.);
[email protected] (A.S.);
[email protected] (A.N.) Department of Environmental Microbiology, Laboratory of Mycology, Faculty of Agrobioengineering, University of Life Sciences in Lublin, 20-069 Lublin, Poland;
[email protected] (J.B.);
[email protected] (T.K.-K.) Department of Plant Physiology, Institute of Biology and Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, 20-033 Lublin, Poland;
[email protected] Correspondence:
[email protected]; Tel.: +48-081-537-59-18
Received: 15 September 2018; Accepted: 14 October 2018; Published: 18 October 2018
Abstract: The endogenous pool of phytoregulators in plant tissues supplied with microbial secondary metabolites may be crucial for the development of winter wheat seedlings during cool springs. The phytohormones may be synthesized by psychrotrophic microorganisms in lower temperatures occurring in a temperate climate. Two fungal isolates from the Spitzbergen soils after the microscopic observations and “the internal transcribed spacer” (ITS) region molecular characterization were identified as Mortierella antarctica (MA DEM7) and Mortierella verticillata (MV DEM32). In order to study the synthesis of indoleacetic acid (IAA) and gibberellic acid (GA), Mortierella strains were grown on media supplemented with precursor of phytohormones tryptophan at 9, 15 ◦ C, and 20 ◦ C for nine days. The highest amount of IAA synthesis was identified in MV DEM32 nine-day-culture at 15 ◦ C with 1.5 mM of tryptophan. At the same temperature (15 ◦ C), the significant promoting effect (about 40% root and shoot fresh weight) of this strain on seedlings was observed. However, only MA DEM-7 had the ACC (1-aminocyclopropane-1-carboxylate) deaminase activity with the highest efficiency at 9 ◦ C and synthesized IAA without tryptophan. Moreover, at the same conditions, the strain was confirmed to possess the strong promoting effect (about 40% root and 24% shoot fresh weight) on seedlings. Both strains synthesized GA in all tested terms and temperatures. The studied Mortierella strains had some important traits that led them to be considered as microbial biofertilizers components, improving plant growth in difficult temperate climates. Keywords: Mortierella; phytohormones; winter wheat seedlings; psychrotrophs
1. Introduction In addition to Penicillium and Aspergillus genera, Mortierella are very common filamentous fungi isolated from the environment [1,2]. The studies on soil ecology describing fungal diversity reported an occurrence of Mortierella strains in soil, rhizosphere, rivers, and lakes on different continents [3–9]. Nagy et al. [1] described Mortierella as widespread in the temperate zone. Most of the strains investigated in detail were isolated from low-temperature environments. The temperature for Int. J. Mol. Sci. 2018, 19, 3218; doi:10.3390/ijms19103218
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psychrophilic microorganisms growth is about 5 ◦ C or below, with the optimum at 15 ◦ C or lower. In the case of psychrotrophs, they grow at a low temperature with the optimum one above 15 ◦ C and maximal growth temperature above 20 ◦ C [10,11]. Winter wheat is one of the most important cereal crops in Europe, mainly sown in fall. In Poland, the optimal seedling season for this plant is at the turn of the second half of September and the first half of October when the average daily temperatures correspond with the optimum growth temperature for psychrotrophs [12,13]. Indoleacetic acid (IAA) and gibberellic acid (GA), as well as ACC-deaminase, are plant growth regulators synthesized by plants as well as a number of soil microorganisms [14]. “IAA is essential for all important processes including seedlings growth. Tryptophan has been identified as a main precursor for IAA biosynthesis pathways in microorganisms (bacteria, fungi) and plants [15]. The dose of Trp recommended for fungal IAA synthesis is from 2 mM to 4 mM [16,17]. Many reports confirmed that even the very small amount of Trp may induce the IAA synthesis [15,18,19]. The term small dose is not defined and the lowest threshold value crucial for auxin synthesis could be strain-dependent.” Among various functions, “GA” may stimulate the synthesis of hydrolytic enzymes activity during the germination of a cereal grain. Phytohormone ethylene plays role in plant development regulation and stress resistance. Moreover, the microbial ACC-deaminase converts the precursor of ethylene (ACC) to the ammonia and α-ketobutyrate and protects plants from excessive concentrations of ethylene [20–22]. The microbial production of phytohormones and phytoregulators, such as ACC-deaminase, is one of the most important direct mechanisms contributing to a rapid and durable soil colonization, which consequently enhances plant growth [23–25]. More attention should be paid to microorganisms promoting plant growth at lower temperatures, as well as in a broad range of temperatures during the selection of microbial components of biofertilizers to be applied in climate conditions of eastern Europe. There are a number of detailed studies on the synthesis of polyunsaturated fatty acids (PUFA) by fungi of the Mortierella genus. However, the literature data lack the information about the production of phytoregulators by Mortierella strains [26–29]. Therefore, in this study, we identified two Mortierella strains growing in a wide temperature range. We have also investigated the synthesis of IAA, GA, and ACC-deaminase and evaluated the effect of these microorganisms on the winter wheat seedlings growth in temperatures characteristic for the temperate zone. 2. Results 2.1. Identification and Optimal Growth Temperature of Fungal Isolates The mycelium of tested Mortierella antarctica (MA DEM7) and Mortierella verticillata (MV DEM32) fungi growing on potato dextrose agar (PDA) formed rosette, cottony, white aerial colonies (Figure 1A,B). Additionally, along with the MV DEM32 mycelium growth, the strain became yellowish. As a result of microscopic observations of both tested microcultures incubated at 20 ◦ C for seven days, we did not obtain fungal sporulation. After seven days incubation at 4 ◦ C, the MV DEM32 isolate formed characteristic sporangiophores with few-spored sporangia (Figure 1C). This strain (MV DEM32) was characterized by the presence of needle-shaped sporangiophores with two to three branches and few-spored sporangia. In order to obtain the sporulation by starvation of Mortierella strains, the attempts were made to culture studied strains on the water agar. However, no growth was observed on this medium. Similarly, no good sporulation of MA DEM7 isolate was noticed. The identification of fungal isolates the internal transcribed spacer (ITS) sequences was investigated and sent to NCBI GenBank. A similarity of 100% to Mortierella antarctica (strain MA DEM7) was detected. The MV DEM32 strain exhibition of 100% homology with Mortierella verticillata Linnem (the synonym for this species is Mortierella marburgensis Linnem) [30–32] was detected. According to the best of our knowledge, this is the first work describing Mortierella verticillata isolated from the
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DEM7) was detected. The MV DEM32 strain exhibition of 100% homology with Mortierella verticillata Int. J. Mol. Sci. 2018, 19, 3218 3 of 17 Linnem (the synonym for this species is Mortierella marburgensis Linnem) [30–32] was detected. According to the best of our knowledge, this is the first work describing Mortierella verticillata isolated Spitzbergen soil. The consensus of the ITS fragment (query) was submitted and deposited in the from the Spitzbergen soil. The consensus of the ITS fragment (query) was submitted and deposited GenBank under the following accession numbers: MH 289781 (strain (strain MA DEM7) and MH 304896 in the GenBank under the following accession numbers: MH 289781 MA DEM7) and MH (strain MV DEM32). 304896 (strain MV DEM32).
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Figure 1. Mycelial growth of (A) M. antarctica DEM7; (B) M. verticillata DEM32 on potato dextrose Figure 1. Mycelial growth of (A) M. antarctica DEM7; (B) M. verticillata DEM32 on potato dextrose agar agar medium. (C) Sporangiophores with few‐spored sporangia M. verticillata DEM32, (PDA)(PDA) medium. (C) Sporangiophores with few-spored sporangia M. verticillata DEM32, “observed in “observed in microcultures on PDA medium, scale bar 50 μm” microcultures on PDA medium, scale bar 50 µm”.
2.2. The Effect of Temperature on the Radial Growth of Mortierella Strains 2.2. The Effect of Temperature on the Radial Growth of Mortierella Strains The ability of two Mortierella strains to grow at five different temperatures was tested, and the The ability of two Mortierella strains to grow at five different temperatures was tested, and the optimal temperature conditions were determined (Figure 2A,B). The experiment was conducted for optimal temperature conditions were determined (Figure 2A,B). The experiment was conducted for six days until the fungal colony “reached the maximum diameter of 9 cm of petri dish”. The results six days until the fungal colony “reached the maximum diameter of 9 cm of petri dish”. The results showed that MA DEM7 and MV DEM32 strains were able to grow at temperature range between showed that MA DEM7 and MV DEM32 strains were able to grow at temperature range between 4 4 and 28 ◦ C. However, the MA DEM32 demonstrated minimal growth at 28 ◦ C (the diameter of colony and 28 °C. However, the MA DEM32 demonstrated minimal growth at 28 °C (the diameter of colony on the the sixth The best best temperature temperature for for mycelial mycelial growth growth of of MA MA on sixth day day of of experiment experiment was was only only 11 cm). cm). The ◦ DEM7 and MV DEM32 was 15 C, which led to the highest growth rates of 1.19 ± 0.08 and 1.35 ± 0.09, DEM7 and MV DEM32 was 15 °C, which led to the highest growth rates of 1.19 ± 0.08 and 1.35 ± 0.09, respectively (Table 1). respectively (Table 1). The Mortierella growth rate at 20 ◦ C between the fifth and sixth day of the incubation was slow, The Mortierella growth rate at 20 °C between the fifth and sixth day of the incubation was slow, as was indicated in our study. At 4 ◦ C, the growth of the MA DEM7 and MV DEM32 colony was as was indicated in our study. At 4 °C, the growth of the MA DEM7 and MV DEM32 colony was observed from beginning of of thethe experiment. TheThe optimal growth temperature for Mortierella strains observed from the the beginning experiment. optimal growth temperature for Mortierella ◦ (MA DEM7 and MV DEM32) on PDA medium oscillated between 15 and 20 C (Figure 2A,B). strains (MA DEM7 and MV DEM32) on PDA medium oscillated between 15 and 20 °C (Figure 2A,B).
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MA DEM 7
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Figure 2. Mycelial growth [cm] at PDA medium measured daily for six days. (A) The diameter of Figure 2. Mycelial growth [cm] at PDA medium measured daily for six days. (A) The diameter of the the M. antarctica DEM7; (B) the diameter of the M. verticillata DEM32 at five temperature conditions. M. antarctica DEM7; (B) the diameter of the M. verticillata DEM32 at five temperature conditions. Values are means of three replicates. Bars represented standard deviations (SD). Values are means of three replicates. Bars represented standard deviations (SD). Table 1. The mycelial growth rate [cm/day] of M. antarctica DEM7 and M verticillata DEM32 at five Table 1. The mycelial growth rate [cm/day] of M. antarctica DEM7 and M verticillata DEM32 at five temperature conditions. temperature conditions. Temperature MA DEM7 MV DEM32
Temperature MA DEM7 MV DEM32 c 4 ◦C 0.77 ± 0.06 c 0.71 ± 0.02 4 °C 0.77 ± 0.06 c 0.71 ± 0.02 c ◦ 9 C 1.0 ± 0.05 b 1.0 ± 0.04 b ◦ a 9 °C 1.0 ± 0.05 b 1.0 ± 0.04 b 15 C 1.19 ± 0.08 1.35 ± 0.09 a ◦ ab 20 15 °C C 1.09 ± 0.05 1.25 ± 0.06 ab 1.19 ± 0.08 a 1.35 ± 0.09 a ◦C d 28 20 °C 0.7 ± 0.02 0.041 ± 0.00 d 1.09 ± 0.05 ab 1.25 ± 0.06 ab Values followed by different letters28 °C are significantly0.7 ± 0.02 d different (one-way0.041 ± 0.00 d analysis of variance (ANOVA) followed by a
Tukey’s post hoc test with the significance evaluated at p < 0.05).
Values followed by different letters are significantly different (one‐way analysis of variance (ANOVA) followed by a Tukey’s post hoc test with the significance evaluated at p
