Enhancing Alfalfa Productivity with Tuber: Associated and Cellulolytic Bacteria in the Aral Sea Region

The work aimed to use biofertilizers to boost alfalfa productivity in the Aral Sea. These “bio” fertilizers contain living bacteria that break down cellulose with the help of cellulases. The object of the research was a year-old alfalfa plant of the variable Agnia variety from the V. R. Williams Research Institute of Feed. Small-scale experiments were conducted at the experimental site of Bolashak University (Kyzylorda city). The studies were conducted in the experimental field of the Department of crop production named after I. A. Stebut of St. Petersburg State Agrarian University in 2012, 2013, and 2014 to identify the most efficient nodule bacteria strains and ascertain their effects on the productivity of alfalfa changeable in the Leningrad region.

Experiments in small land cells were conducted in several configurations in the agro-biotechnological field of Bolashak University in Kyzylorda. Alfalfa nodule bacteria strains and their mixtures with cellulolytic bacteria and mineral fertilizers were employed to create different variants. For seed inoculation, the following strains of nodule Sinorhizobium meliloti bacteria were used: the production strain Sinorhizobium meliloti 415b, the promising strains Sinorhizobium meliloti IMVL5-16 and IM VL 5 (Microbiology), No. 4 W (CLB+ Sinorhizobium meliloti IMVL5-1), and ammonium nitrate was used as a mineral fertilizer for alfalfa. In the control variant, no nodule bacteria treatment was applied to the alfalfa seeds. Alfalfa seeds of the “Semirechensk local” type was sown in the cells.

The strains were cultivated on the Luria Bertani (LB) medium at 37℃. Alfalfa seedlings were surface sterilized for 5 minutes with 70% ethanol and then washed with sterile water. The seeds were sown on moist filter paper in Petri dishes and allowed to sprout in a darkened growth environment. The seeds from the previous 20 years must be scarified so that the outer shell is broken before they can be utilized for sowing. To disrupt the integrity of the seeds' outer shell, the seeds were first treated with a biological preparation of CLB before being deposited for five hours. Then the seedlings were injected with the experimental strains, whereas seedlings acting as a control variety were not inoculated. After following this procedure, the sowing was performed.

Productivity analysis was used to gauge how well nodule bacteria were performing. For each cell, the number of plants in the 1 m² of soil was calculated. The degree of acetylene-reducing ability of root nodules was used to calculate nitrogen-fixing (nitrogenase) activity, which was represented in micromoles of ethylene produced by one plant's nodules every hour. Roots with nodules were put in glass vials, and 10 percent acetylene was introduced via a rubber membrane after the air was drawn out. One sample required one hour of incubation. An Agilent Technologies 6855 Network GC System (USA) gas chromatograph was used to examine the gas mixture.

During the budding stage, the number of photosynthetic pigments was measured. Five randomly chosen plants of one variant's middle layer of leaves were chosen. The level of photosynthesis was assessed using the following formula. The plant-filled jar was put in a hermetically sealed space that was being blasted with atmospheric air at a rate of 15 L/min. One L/min of gas was taken into the gas analyzer at the chamber's output. Through a water filter, a KG-2000 incandescent bulb was used to illuminate the chamber. After recording the rate of CO₂ uptake by entire plants, the chamber was opened, the plants were cut, and then it was closed once more to measure the rate of soil respiration with roots. Using the Lowry technique in the budding phase, the protein content of alfalfa leaves was evaluated; the quantitative and qualitative contents of amino acids were assessed using ion-exchange liquid-column chromatography and an automated analyzer.

The choice of specific measurements in the study was made with the intention of comprehensively assessing the effects of the treatments on alfalfa productivity. Each chosen measurement reflects a different aspect of the plant's performance, providing insights into its physiological, biochemical, and metabolic responses to the applied treatments. These chosen measurements collectively provide a comprehensive understanding of the treatment effects on alfalfa productivity. Nitrogenase activity directly addresses the plant's nitrogen supply, photosynthetic pigments give insights into energy capture and utilization, and protein content relates to the nutritional value of the plant. By examining these aspects, the study can holistically evaluate the efficiency of the applied biofertilizers, bacteria strains, and mineral fertilizers in enhancing various facets of alfalfa growth, making informed conclusions about their potential to boost overall productivity.

The implementation of nodule bacteria-based preparations is an eco-friendly method to boost crop yields and enhance soil fertility. Numerous studies show a positive correlation between biologics utilization and enhanced plant growth and development [15-17]. Given its superior value (digestibility and protein content), widespread cultivation, inexpensive cost of cultivation, and seasonal distribution, alfalfa is among the most significant feed legume crop [8]. However, its growth and development are heavily influenced by cultivation conditions, such as water supply, temperature, pathogen infection, and soil contamination [9-11, 18-20]. The present study demonstrated that seed treatment with a preparation containing CLB and Sinorhizobium meliloti strains significantly improved the green mass yield and height of alfalfa plants. These data confirm the findings presented in previous research. Thus, a recent study evaluated the development and nodulation of alfalfa plants in conjunction with six different strains of Sinorhizobium meliloti under water-scarce conditions [8]. The results showed that compared to uninoculated plants, Sinorhizobium meliloty treatment improved the plants' ability to cope with stress. Therefore, in comparison to the non-inoculated control in all water stress levels, the plants that received Sinorhizobium meliloti inoculation increased shoot growth, root length, and shoot and root dry weights.

According to the findings of another investigation, the control variant's nitrogen content and alfalfa dry biomass were considerably lower than those produced by the Sinorhizobium meliloty inoculation treatment [9]. The present study showed that variant No. 4 Zh (CLB+ Sinorhizobium meliloti IM VL 5-1) treatment produced roughly the same outcome as the studies discussed, increasing alfalfa dry mass and plant growth. Alfalfa grown on soil polluted with zinc benefited from Sinorhizobium meliloty inoculation treatment because it produced less toxic hydrogen peroxide and malondialdehyde, which improved the crop's quantity, quality, and safety for consumption [7]. Similarly, Sinorhizobium inoculation reduced copper and zinc stress in alfalfa plants, which resulted in greater root and above-ground plant length and dry weight compared to the control [10, 21]. The putative ability of biologicals to remediate the soils of the Kyzylorda region was out of the scope of the present study.

However, the studied strains show the promise to increase soil fertility in the Aral Sea region, and future research could focus on the possibility to implement these findings into agricultural practices. It was shown that under limited phosphorus availability, compared to uninoculated plants, the treatment of alfalfa plants with Sinorhizobium alone or in combination with Pseudomonas dramatically increased plant growth, as well as physiological parameters, and biochemical features [11, 22]. These results were confirmed in the present study as Sinorhizobium treatment of alfalfa generally enhanced the crop’s morphological indicators in all variants except the control. Finally, Sinorhizobium-inoculated alfalfa plants also showed higher alkali resistance, higher levels of chlorophyll, comparatively longer roots, greater plant height, and more branches, according to Xing et al. [12]. Similarly, the present results indicated increased photosynthesis levels in Sinorhizobium-treated crops in all variants but the control.

According to the data obtained in the present study, cellulose-degrading bacteria also contribute to the increased alfalfa yield. These results find endorsement in literature as well, as cellulases produced by these microorganisms have vast agro-technological implementations [23, 24]. It was demonstrated that adding cellulases to crops could improve plant development, including improved seed germination and protective benefits. They are thought to primarily act as biocontrol agents that facilitate disease inhibition and/or activate plant defense systems [25, 26]. The precise relationship between these enzymes and plants has not yet been fully understood. Similarly, in the present study, the exact mechanism of cellulose-degrading bacteria’s effect on alfalfa growth and development is not determined. A hypothesis is made that celluloses produced by the studied stains act as a scarification agent facilitating seed germination.

However, cell expansion, seed germination, and seed protection are only a few biological processes that cellulases appear to be engaged in that might be connected to plant development. According to the literature, plants must use hydrolytic enzymes like cellulases to weaken their cell walls in order to develop properly [23]. These findings can be illustrated with the example of coffee seed germination. Coffee tree seeds take a long time to germinate because they are sown immediately after the harvest of the crop which falls during a cold season, besides the fact that parchment acts as a barrier to prevent the seed from absorbing water [27, 28]. Coffee seeds from the cultivar “Acaiá do Cerrado” were soaked in cellulase solution at various doses in an experiment to determine the impact of exogenous administration of the enzyme on coffee seed germination. When the seeds were exposed to the solution for 144 hours, it was discovered that the cellulase enzyme offered the highest index of speed germination and the highest germination percentage [27]. Another study showed that during the germination of lettuce seeds, cellulase contributes to the weakening of the endosperm cap and the extension of the radicle [29, 30]. The present study showed that the implementation of cellulose-degrading bacteria improved plant growth, however, additional research is needed to link the obtained results with the cellulase impact on seeds’ integrity.

Additionally, seeds that had been pretreated with cellulases showed improved resistance. For example, cellulases may have direct impacts on P. splendens, possibly by releasing glucans that might serve as plant endogenous elicitors or by releasing poisonous compounds from the seeds that prevented P. splendens from germinating [23]. Given that cellulose is a key component of pathogenic oomycetes cell walls, cellulases seem to be crucial in their antagonistic interactions [23]. The present research implemented bacterial strains only. However, numerous cellulolytic fungi, in addition to bacteria, are known to play a crucial role in agriculture by promoting improved seed germination, quick plant growth and flowering, superior root systems, and higher crop yields [31-33]. These fungi include Trichoderma sp., Geocladium sp., Chaetomium sp., and Penicillium sp. Despite the fact that these fungi affect plants directly (perhaps through growth-promoting diffusible substances) and indirectly (by reducing plant infections and diseases), it is not yet known how these fungi support greater plant performance [31, 33]. Future studies regarding the effect of cellulose-degrading microorganisms on alfalfa growth in the Aral Sea region should focus not only on the crop parameters but on the mechanisms of cellulases interplay with seeds’ outer layers and putative antagonistic interactions with soil microbiota. It would be also beneficial to study cellulose-degrading fungi for possible implementation in agricultural practices.

Thus, the positive effect of the preparation containing cellulose-degrading bacteria and Sinorhizobium meliloty on alfalfa crops can be attributed to the symbiotic interactions between these microorganisms and the plant. Cellulase-producing bacteria putatively enhance alfalfa seed germination by weakening the outer layers of the seeds and promoting their growth. Additionally, cellulases play an important role in bacterial antagonistic interactions and protect the alfalfa seeds and plants from pathogenic microorganisms. Last but not least, cellulases degrade the organic matter and enrich the soil with elements crucial for alfalfa development.

The research underscores the need for further investigations to uncover the intricate interplay between microorganisms and plants, particularly regarding cellulase interactions with seeds and potential antagonistic effects on soil microbiota, ultimately enhancing our understanding of these processes for agricultural applications. The positive correlation between Sinorhizobium meliloty treatment and alfalfa green mass yield is most likely due to the symbiosis between the strain and the host plant. It is highly likely that improved nitrogen fixation, owing to Sinorhizobium meliloty penetration into the alfalfa legumes, boosts the plant’s metabolism and enables increased resistance to harsh growth conditions. Future studies could elaborate on the exact mechanisms of plant-bacterial interactions between alfalfa and microorganisms in the preparation studied.