Study of the potential of Pseudomonas Protegens A-CMC-05 and Gordonia Paraffinivorans A-CMC-11 strains for use in agricultural biotechnology

Excessive use of chemical fertilizers has led to various negative consequences such as accumulation of harmful elements in the soil, groundwater pollution, reduction of soil organic matter content and fertility, and deterioration of soil physical and chemical properties. In recent years, special attention has been paid to the industrial potential of microorganisms for use as biological fertilizers, their ability to improve nutrient availability, enhance plant growth and productivity, and protect the environment from negative impacts. A large group of soil bacteria has been shown to have a positive effect on plant growth, which is associated with such properties as increased availability of mineral nutrition elements for plants, production of metabolites with hormonal and signaling functions (auxins, cytokinins, gibberellins, abscisic, salicylic and jasmonic acids), induction of mechanisms of systemic resistance to stresses of abiotic and biotic nature. Bacillus and Pseudomonas are among the most studied and widely used producers of bacterial enzymes and destructors of organic compounds, as well as plant growth-promoting bacteria and antagonists of plant pathogens. In the present study, the growth-stimulating and bioprotective properties of strains Pseudomonas protegens A-CMC-05 and Gordonia paraffinivorans A-CMC-11, which we had isolated earlier from municipal wastewater treatment plants in Surabaya, Indonesia, were investigated. The representatives of Pseudomonas and Gordonia are widely distributed in nature and are actively studied due to their ability to destruct, transform and synthesize organic compounds. P. protegens is known as a plant growth-promoting rhizobacterium. No published data were found for G. paraffmivorans to investigate their potential as plant growth stimulators or biocontrol properties, but such works are available for other members of the genus Gordonia. The inhibitory activity of strains P. protegens A-CMC-05 and G. paraffinivorans A-CMC-11 against the phytopathogenic fungus of the genus Fusarium, representatives of which are often associated with severe crown and root rot diseases of wheat, was investigated. When Fusarium equiseti D1 was co-cultured in the presence of each of the bacterial strains, changes in the morphology of the fungal mycelium and inhibition of the growth zone were observed (See Fig. 1А). When the fungal fragment was inoculated onto freshly grown bacterial turf, the inhibitory activity of P. protegens A-CMC-05 was as prominent, whereas strain G. paraffinivorans A-CMC-11 showed less pronounced antagonism (see Fig. 1B). In the third cultivation variant, which consisted of inoculating the fungal mycelium into the center of the Petri dish at the same distance (3.5 cm) between two bacterial growth zones (0.7-0.8 mm in diameter), in the presence of the Pseudomonas sp. strain A-CMC-05, a more noticeable inhibition of mycelial growth was also observed than in the presence of the Gordonia sp. strain A-CMC-11 (See Fig. 1С). Inhibition of radial mycelial growth ranged from 11% to 13% for strain A-CMC-11 and from 36% to 67% for strain A-CMC-05. In addition to inhibition of the growth zone, strain A-CMC-05 inhibited the development of aerial hyphae of Fusarium equiseti D1, only weak growth in the depth of the agarized medium was observed (See Table 1). An experiment was conducted to study the growth-stimulating properties promoting rhizogenesis of unrooted explants of barberry of Thunberg (Berberis thunbergii Aurea), selected as an object for research (See Fig. 2). As a result, rhizogenesis processes were observed in 12.1% of explants planted in sterile soil treated with the culture liquid solution of strain A-CM-05. In the experiment with strain A-CM-11, root formation was detected in 7.5% of explants. In the control experiment with the addition of the Kornevin commercial preparation based on 4(indol-3yl)butyric acid (IBA) the efficiency of rhizogenesis was 15%. Analysis of the obtained results indicates that strain A-CMS-05 has pronounced properties promoting rhizogenesis in barberry plants used as an object of research and its efficiency in stimulating rhizogenesis is comparable to the efficiency of the preparation based on IBA. In subsequent experiments, in addition to the effect of strains A-CMC-05 and A-CMC-11 individually, their effect on plant growth and rhizogenesis in the consortium was investigated. In experiments with germination of wheat (Triticum aestivum L.) seeds in sterile soil, strains of G. paraffinivorans A-CMC-11 and P. protegens A-CMC-05, including as part of a consortium, had no effect on seed germinability (See Fig. 3А). At the same time, the formation of a denser earth clod at the roots of wheat seedlings in the experiments with each of the strains and as part of the consortium compared to the control was noted (See Fig. 3B). Statistically significant differences were also revealed between the lengths of seedlings when seeds were treated with G. paraffinivorans A-CMC-11 and the lengths of wheat roots under the influence of P. protegens A-CMC-05 (See Fig. 4). The use of strains as part of a consortium showed no significant results. Additionally, the effect of bacterial strains and consortium on rooting efficiency and shoot length in raspberry (Rubus ideus L.) explants of “Cassiopeia” variety was investigated (See Fig. 5). In contrast to the experiment with barberry explants, no positive effect of individual strains or their consortium on rhizogenesis processes in raspberry was found. Moreover, the efficiency of root formation by the number of rooted explants was the most significant in the negative control without any soil or explants treatment and amounted to 87% (See Fig. 5A). We did not analyze root and shoot masses except for visual assessment. The total root mass in experimental and control experiments could differ and, among other things, affect the average shoot mass, which visually increased after treatment with culture liquid of the strains and consortium due to a wider leaf plate (See Fig. 6). In addition, treatment of explants with the culture fluid of P. protegens A-CMC-05 increased the average shoot length by 40% compared with the negative control and by 73% compared with the positive control (See Fig. 5B). The studies demonstrated the potential of P. protegens A-CMC-05 and G. paraffinivorans A-CMC-11 as biocontrol agents and plant growth stimulators. P. protegens A-CMC-05 showed more pronounced results and is a more promising agent for further studies of growth-stimulating and bioprotective properties. The article contains 6 Figures, 1 Table, 40 References. The Authors declare no conflict of interest.

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