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Article

Ensuring Tree Protection, Growth and Sustainability by Microbial Isolates

by
Lenar R. Valiullin
1,2,3,
Ascar R. Gibadullin
1,
Vladislav I. Egorov
2,
Rinat S. Mukhammadiev
1,
Rishat S. Mukhammadiev
1,
Vladimir V. Sakhnov
4,
Rupesh Kumar Singh
5,6,
Svetlana N. Sushkova
7,
Svetlana V. Kozmenko
7,
Tatiana M. Minkina
7,
Vishnu D. Rajput
7,
Anuj Ranjan
7,
Inna V. Zamulina
7,
Mikhail G. Baryshev
3,
Mikhail A. Sevostyanov
3,8,
Nikolai I. Budynkov
3,
Larisa L. Sviridova
3,
Saglara S. Mandzhieva
7,
Valery P. Kalinitchenko
3,9,* and
Vladimir I. Cherniavskih
10
1
Department of Biotechnology, Federal Center for Toxicological Radiation and Biological Safety, 420075 Kazan, Russia
2
Department of Forestry and Ecology, Kazan State Agrarian University, 420015 Kazan, Russia
3
Department of Epidemiology and Phytosanitary, All-Russia Phytopatology Research Institute, 143050 Odintsovo, Russia
4
Department of Forest Crops, All-Russian Research Institute of Silviculture and Mechanization of Forestry, 141202 Pushkino, Russia
5
Centre for the Research and Technology of Agro-Environmental and Biological Sciences, University of Tras-O-Montes and Alto, 5000-801 Vila Real, Portugal
6
Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
7
Academy of Biology and Biotechnologies, Southern Federal University, 194/1 Stachki Prosp., 344090 Rostov-on-Don, Russia
8
Department of Composite Materials, Institute of Metallurgy and Materials Science Named after A.A. Baikov, 119334 Moscow, Russia
9
Sustainability Department, Institute of Fertility of Soils of South Russia, 346493 Persianovka, Russia
10
Forage Crop Selection Center, Federal Williams Research Center of Forage Production and Agroecology, 141055 Lobnya, Russia
*
Author to whom correspondence should be addressed.
Sustainability 2024, 16(17), 7837; https://doi.org/10.3390/su16177837
Submission received: 30 March 2024 / Revised: 12 August 2024 / Accepted: 29 August 2024 / Published: 9 September 2024
(This article belongs to the Section Sustainable Forestry)
Browse Figures
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Abstract

:
Antimicrobial properties of the new strains of micro-organisms isolated from natural sources of various ecological niches in the Moscow region and the Republic of Tatarstan were studied. Antifungal activity of isolates was detected in a test culture of toxin-producing microscopic fungi that can cause animal and plant diseases: Aspergillus flavus, Candida albicans, Fusarium oxysporum and Penicillium spp. Of the 46 studied micro-organisms of genera Bacillus, Lactobacillus, Lactococcus and Streptomyces isolates, there are four strains (Bacillus subtilis, Lactobacillus plantarum, Propionibacterium freudenreichii and Streptomyces spp.) that showed an ability to produce biologically active metabolites with a pronounced antimicrobial potential against phytopathogenic fungi metabolites. Based on the selected four strains, a Bacterial product LRV composition has been created. Scots pine, pedunculate oak and small-leaved linden seedlings with single and double foliar treatment and Bacterial product LRV at a concentration of 10 mL/L led to an increase in the growth of the aboveground part by 31.8, 51.9 and 25.4%, respectively, and the underground part by 25.0, 37.2 and 25.7%, respectively, compared to the control. The weight of seedlings at the end of the study exceeded the control variant by an average of 26.0, 44.0 and 78.0%, respectively. Plant protection Bacterial product LRV use did not have a significant effect on the group of molds that caused the powdery mildew and Schütte disease damage to trees. The Biological product LRV provided plant protection from fungal diseases caused by Lophodermium pinastri Chev. and Microsphaera alphitoides.

1. Introduction

Currently, violations in forest cultivation have a negative impact on the climate and cause a land anthropogenic disturbance [1,2,3]. Ensuring the land restoration and biological resources conservation, forests provide human well-being. Forest and agricultural land restoration ensures sustainable ecosystem development. It is an important tool in land management, increasing the biodiversity and soil carbon content, preventing pollutants from uncontrolled transport and providing CO2 sequestration [4,5,6,7,8]. The tree diseases strongly limit the success of silviculture. Bacteria are important biological control agents of forest plant diseases [9,10]. Forest restoration ensures economic and recreational social development [11,12,13,14].
In Russia, the reforestation and forest improvement are underway. The State program “Development of forestry in the Tatarstan Republic in 2014–2024”, launched in 2013 [15], was focused on the restoration of felled trees and an improvement of thinned plantings, forest zones and forest park facilities. The program’s purposes were forest fire detection and extinguishing, forest protection from harmful organisms and adverse factors, forest high-quality use and forest protection and reproduction. Modern advances in the development of plant protection products have been achieved almost exclusively in the chemical industry. Now, chemical plant protection products are the main means of combating plant and forest phytopathogens in the world [1,16,17].
Many authors have shown that long-term use of agrochemicals is associated with a persistent level of environmental pollution, causing an environmental risk in biological systems [3,4,18]. Long-term exposure to synthetic fungicides has led to a decrease in application efficiency due to the development of resistance mechanisms in plant pathogens [2]. This has led to an increased use of chemicals, with consequent accumulation of residues in agricultural products and their by-products, which in turn have harmful effects on both human and animal health [3]. A promising alternative to standard pest management is a biological control approach based on the natural antagonistic effects of biological agents to mitigate the harmful effects of plant pathogens [4].
The biological control mechanisms carried out by the biological agents are diverse and depend on the specific features of both the pathogen and the antagonist, as well as on their density and the specificity of the interactions that occur between them [5,6]. Successful biological control is usually characterized by an activation of multiple mechanisms and targets synergistically aimed at controlling a pathogen and/or its deleterious effects on the biological targets [7,8]. According to technological standards, the biopesticides or bioprotection products must be non-toxic for humans and animals and for the environment [11,12,13]. A direct antagonism (e.g., hyperparasitism and predation) occurs when there is a very high affinity between a pathogen and its antagonist [14,19]. The existing pathogen suppression mechanisms include the production of volatiles, antibiotics and other secondary metabolites of a microbial life cycle [20,21,22]. The production of volatile organic compounds is of ever-increasing interest in the scientific community due to the various benefits of their application [23].
The aim of the study was the extraction of isolates of micro-organism genera from natural sources in various ecological niches in the Moscow region and in the Republic of Tatarstan. Moreover, we sought the synthesis of a biological product based on the newly isolated microbial strains, stimulating seedling protection from fungi diseases caused by Lophodermium pinastri Chev. and Microsphaera alphitoides.

2. Materials and Methods

2.1. Region of Investigations

The research was carried out in the Kaibitsky afforestation enterprise of the Republic of Tatarstan. The Kaibitsky forest area belongs to the Volga region.
The Volga region is a relatively humid territory. However, precipitation is distributed unevenly throughout the territory. In an elevated part of region adjacent to the Volga River, rainfall is higher than 450 mm/year. Most precipitation (about 70%) falls during a warm period from April to October [24].

2.2. Experiment Location, Conditions and Layout

In the Berlibash subdivision of Kaibitsky afforestation enterprise located on the Berl river (55°19′17″ N 48°06′28″ E), a development of the Scots pine (Pinus sylvestris), pedunculate oak (Quercus robur) and small-leaved linden (Tilia cordata) seedlings under a Bacterial product LRV application were studied. The Bacterial product LRV has been prepared on the basis of bacterial isolate association to provide the seedlings growth stimulation and protection from phytopathogens. The origin of Bacterial product LRV, its properties and a test in the controlled environment conditions showed the effectiveness of Bacterial product LRV, as presented in detail below in the text. The experiment was carried out in triplicate.
The total experimental area was 200 m2. An individual experimental plot-accounting area was 7 m2. The experiment options of a biological preparation application were as follows:
1. Control—seedlings sprayed with water once;
1a. Control—seedlings sprayed with water twice;
2. Experimental group 1—seedlings treated with the Bacterial product LRV at a dose of 10 mL/L once;
2a. Experimental group 2—seedlings treated with the Bacterial product LRV at a dose of 10 mL/L twice;
3. Experimental group 3—seedlings treated with the Bacterial product LRV at a dose of 4 mL/L once;
3a. Experimental group 4—seedlings treated with the Bacterial product LRV at a dose of 4 mL/L twice.
The accounting experimental plot site area of 3-row seedling strips was allocated by protective 2-row seedling strips.
A Bacterial product LRV solution spraying dose was 100 mL/m2. A foliar treatment of seedlings with Bacterial product LRV was carried out after the seedlings bloomed. A working solution was prepared immediately before the application. The seedling plots treated with 100 mL/m2 water served as control. A seedling re-treatment was carried out a month after the first treatment. At the end of the growing season, the seedlings number was counted in each repetition of the experiment.
Fifty seedlings were selected from each variant at the end of September. The seedling aerial and underground parts were separated. The seedling height, root length and aerial and underground parts per year of growth were measured for each seedling. The seedling root and aerial part dry weight was determined after oven drying at a temperature of 105 °C.
Samples for a Bacterial product LRV synthesis taken from various natural sources in the Moscow region and the Republic of Tatarstan served as a material for obtaining micro-organism cultures. For selection, the micro-organisms were cultivated on various liquid (MPA, Sabouraud broth) and dense (MPA, wort agar, Sabouraud agar, MRS, M9) nutrient media with a subsequent study of their morphological, tinctorial and biological properties [25,26,27,28,29].
An isolate antifungal activity was established in a toxin-producing microscopic fungi test culture that causes various plant diseases: Fusarium oxysporum, Aspergillus flavus, Penicillium spp. and Candida albicans—a yeast also pathogenic for humans (Collection of Micro-organisms of the All-Russian Research Institute of Phytopathology, Moscow region, Russia). The bacterial and test isolates were cultivated at a temperature of 37 ± 1 °C and 28 ± 2 °C, respectively, in a test tube on a slant agar of the following composition (%): glucose—0.63, enzymatic peptone—2.1, sodium chloride—0.65, sodium hydrogen phosphate—0.35, potassium dihydroorthophosphate—0.06 and microbiological agar—0.12 [30].
The antagonistic activity of isolates of micro-organisms against microscopic fungi was determined using a plate method (double culture method) [31]. To obtain an isolate sample, individual bacterial isolate was cultivated in a Petri dish on a Luria–Bertani (LB) agar medium at 37 ± 1 °C for 48 h to the moment of a continuous micro-organism mycelium cover formation. The fungal colonies were previously grown in another Petri dish on a Czapek–Dox agar medium at 28–30 °C for 7 days till the continuous microscopic fungi cover surface formation. Then, the test micro-organism culture mycelium discs 6 mm in diameter were cut out from the micro-organism mycelium cover on the LB agar medium surface. These blocks with a test culture were transferred to another Petri dish and placed on the surface of a fungal colony on the Czapek’s agar medium. The disks with a mycelium of test isolate were evenly distributed at a distance 3 cm from one another and from the edge of Petri dish with a fungal colony on the Czapek’s agar medium. As a control, fungi cultures without texted bacterial strain application were used. An incubation of micro-organisms was carried out at 28–30 °C for 5 days. After cultivation, the maximum and minimum size of the pathogen growth inhibition zone was measured.
Lactic acid micro-organism isolate antifungal activity was also assessed by a counter cultures method [16]. For this, mycelium disks (diameter 6 mm) were cut out from a fungal colony previously grown on Czapek–Dox agar for 7 days at a temperature of 28–30 °C and placed in the center of MRS-SA agar plates (without sodium acetate and ammonium citrate). The studied isolates inoculated with a specified medium, Czapek–Dox agar, were placed at a distance of 2 cm from an agar plate edge. As a control, the plates with disks of fungi without bacterial strains were used. Micro-organisms were incubated at 28–30 °C for 120 h.
The antifungal properties of the biologically active metabolites of isolated micro-organisms in relation to pathogens of various diseases of agricultural plants were determined by the method described in [32,33,34,35,36,37].
Identification of isolated micro-organisms was carried out on the basis of morphological–cultural and physiological–biochemical properties, guided by the determinants of Bergey and Kaufman [38,39,40]. A primary identification of the strains was carried out by studying their tinctorial properties using the Gram stain method [41]. The morphological features of micro-organism cells were studied by obtaining live and fixed stained preparations using a bright field and phase contrast microscopy [41]. A studied bacteria motility was determined in a “crushed drop” and a “hanging drop” preparations from the daily cultures with their further microscopy.
The ability of isolates to grow in the agar media was assessed by cultivating micro-organisms for two days at a temperature of 10–50 °C and a pH of 5.0–8.0 [41]. Enzymatic activity w determined and carbohydrate fermentation tests were conducted using the generally accepted methods [42,43]. A fermentation of sugars by isolates was studied using the “variegated series” method. Glucose, lactose, galactose, assucrose, maltose, fructose, rhamnose, arabinose and sorbitol were used as carbohydrates. The micro-organisms were seeded in a liquid nutrient medium of 1% substrate (carbohydrate) and bromcresol purple dye at a concentration of 0.03 mg/mL. A microorganism ability in carbohydrates fermentation was tested accounting a medium discoloration (from purple to yellow).
To study a selected isolate’s enzyme activity, the bacterial cultures were grown on agar-modified corn–lactose and MRS media, as well as on a synthetic medium containing (g/l) sodium citrate—1.29; (NH4)2HPO4—4.75; K2HPO4—9.6; MgSO4 × 7H2O—0.18 (pH 7.0 ± 0.2) [44,45,46,47]. Carboxymethylcellulose (CMC), water-soluble starch and casein at a concentration of 1.0% were used as a carbon and nitrogen source. Olive oil, in doses of 20, 40, 60 and 80, was added to the medium at a concentration of 0.5%. Bacteria were cultivated at 37 ± 1 °C until hydrolysis zones appeared around their colonies, which were used to judge the ability of isolates to produce hydrolytic enzymes.
The effect of Bacterial product LRV on a Scots pine biennial seedlings infected with Schutte’s disease and a pedunculate oak biennial seedlings damaged by powdery mildew was studied. Both Schutte’s disease and powdery mildew are caused by the group of molds.

2.3. Sampling and Analyzes

The soils, fermented dairy products, raw cow’s milk, farm bird and animal gastrointestinal tract and feces contents, as well as agricultural crop samples, were collected in the Moscow region and the Republic of Tatarstan.
Material for the experiment was collected in every studied environment in the following quantities: soils 2 kg, fermented dairy products 2 L, raw cow’s milk 2 L, farm bird droppings 1 kg, animal gastrointestinal tract contents 0.8 kg, feces 0.8 kg, agricultural crops 3 kg. In experiment, 60 g of material was used for the extraction.
A pH value was measured using a potentiometric pH meter during a water-soluble salt extraction. The ratio of micro-organism inoculate and distilled water was 1:2 [48].
Activity of antagonistic micro-organisms against microscopic fungi was determined using the Methodological Instructions MU 2.3.2.2789—10. The microbiological studies were carried out in compliance with the sanitary rules for a safe operation with the micro-organisms of III–IV pathogenicity groups [49].

2.4. Data Processing and Statistical Analysis

The experimental data were statistically processed in the Jamovi software 2.4.14 environment. Tukey’s post hoc test was used for comparison differences between groups. An assessment of the results’ reliability was conducted. The level of significance was p ˂ 0.05 [50,51]. The average values (M) and standard errors (m) were calculated.

3. Results

From the samples of soil, raw cow’s milk, fermented milk products, gastrointestinal tract contents, farm bird droppings, animal feces and crops collected in the Moscow region and the Republic of Tatarstan samples, 46 micro-organism isolates from various taxonomic groups were taken. The study of phenotypic characteristics of the isolated strains, including cell morphology, colony shape and color, acid production, growth pattern, Gram stain and other features, were studied. The results showed the genetic diversity in micro-organisms. The active growth of bacteria on selective media was noted from the first day of the cultivation. Among isolated micro-organisms, most belonged to the bacteria of genera Bacillus, Lactobacillus, Lactococcus and Streptomyces.
Of the 46 studied micro-organisms, 4 strains (Bacillus spp., Propionibacterium spp., Lactobacillus spp., Streptomyces spp.) had a pronounced ability to inhibit the growth and development of the various groups of microscopic fungi. A micro-organism’s antagonistic activity against microscopic fungi is presented in Table 1.
The selected micro-organism strains of the micro-organism genera presented in Table 1 with an antimicrobial potential were classified corresponding to determinants “Bergey” and “Kaufman” as Bacillus subtilis, Lactobacillus plantarum, Propionibacterium freudenreichii and Streptomyces spp. species (Table 2).
For a more detailed characterization of metabolic features of selected cultures of micro-organisms, optimal temperature and pH for growth, as well as the possibility of using various substrates for metabolism, were assessed (Table 2). The isolates were grown at a temperature from 9 to 46 °C and a pH from 3.0 to 9.0 as limits of cultivation. Some strains had an acid-forming activity and an ability to grow in a media with carbon-containing compounds. The bacteria fermented various carbohydrates, such as fructose, maltose, glucose, galactose, mannitol, sorbitol, mannose, sucrose and many other sugars, indicating their ability to use various organic carbon compounds for their metabolism. The strains had the ability to produce amylase, protease, cellulase and lipase enzymes. The most active producers of hydrolases were B. subtilis and L. plantarum strains.
Thus, out of 46 micro-organisms isolated, 4 strains were selected, namely Bacillus subtilis, Propionibacterium freudenreichii, Lactobacillus plantarum and Streptomyces, capable of producing biologically active substances with a pronounced antimicrobial effect. Of isolated micro-organisms with antimicrobial activity, the strains of B. subtilis and L. plantarum are of the greatest interest for studying the properties of hydrolases. The results obtained open up the possibility of using the micro-organisms selected by us and their metabolites as a protective agent or a stimulating additive to agronomic biological preparations.
Table 3 presents some biometric indicators of the seedlings before treatment with Bacterial product LRV at the Berlibash subdivision of the Kaibitskoye afforestation enterprise.
The Scots pine, pedunculate oak and small-leaved linden biennial seedlings biometric indicators obtained in the Berlibash subdivision of Kaibitsky afforestation enterprise are presented in Table 4.
For pedunculate oak biennial seedlings, maximum growth and development were observed when the Bacterial product LRV was sprayed twice at a dose of 10 mL/L. A seedling weight in this variant exceeds the control variant by 26%. With a double application of the Bacterial product LRV at a dose of 4 mL/L, the weight of a seedling exceeds the control by 24%. In these variants, the seedlings’ biometric indicators are significantly ahead of those in other variants of the experiment.
A double foliar treatment of the small-leaved linden biennial seedlings with Bacterial product LRV at a dose of 10 mL/L provided an increase in plants’ aboveground-part growth by 51.9% and underground-part growth by 37.2% compared with the control. The total weight of seedlings at the end of the study exceeds the control variant by an average of 44.0%.
In Scots pine biennial seedlings, the greatest increase in biometric parameters was shown in the variant with a double application of the Bacterial product LRV at a dose of 10 mL/L. A double Scots pine foliar spraying with the Bacterial product LRV at the indicated dose led to an increase in plant aboveground-part growth by an average of 25.7% and underground-part growth by 25.4% compared with the control. Accordingly, an increase of 3.3% and 3.1%, respectively, in the accumulation of seedling shoot and root biomass was observed.
An important object of the study was a Bacterial product LRV effect on fungi caused forest species disease.
Figure 1 and Figure 2 show the Bacterial product LRV effect on the growth and development of the aboveground part of seedlings of pedunculate oak and small-leaved linden in the Kaibitsky afforestation enterprise.
We studied a Bacterial product LRV effect on a Scots pine biennial seedlings infection with Schutte’s disease (Table 5).
As can be seen from Table 5, the Bacterial product LRV did not have a pronounced effect on the spread of Scots pine Schutte’s disease (Figure 3).
We studied a Bacterial product LRV effect on the powdery mildew of pedunculate oak biennial seedlings in the Kaibitsky afforestation enterprise (Table 6).
Table 6 data show an insignificant effect of the application of Bacterial product LRV at different doses on the powdery mildew spread in pedunculate oak seedlings. A value of this indicator of 72.0–79.0% was at a control option level. At the same time, a Bacterial product LRV treatment with doses of 10 mL/L, 50 mL/L and 100 mL/L had a pronounced positive effect on the development of aerial and underground parts of the pedunculate oak and small-leaved linden seedlings. A Bacterial product LRV effect at a dose of 50 mL/L on an aerial part of pedunculate oak seedlings is shown in Figure 4.
The results obtained during the study indicate a Bacterial product LRV potential in the young seedlings’ growth and development.

4. Discussion

The environmental and economic importance of Scots pine, pedunculate oak and small-leaved linden is considerable. However, the morphological, biological and ecological features of these species determine their susceptibility to fungi diseases caused by Lophodermium pinastri Chev. and Microsphaera alphitoides. In consequence, the content and toxicogenicity of phytopathogenic micro-organisms and the degradation of a stand and the soil increased [17,18,52,53]. In this regard, it is relevant to search for new ways to increase forest crop seedling productivity while minimizing an adverse environmental impact. Thus, new, improved measures at higher biological and technological levels are required for the protection and reproduction of Scots pine, pedunculate oak and small-leaved linden [54,55,56].
New environmentally friendly developments, reducing the degree of the negative impact of biotic and anthropogenic stress factors on the trees and soil, are needed. A number of publications have shown that highly effective and environmentally friendly natural growth regulators make it possible to stimulate the growth and development of plants and increase the standard planting material yield and its resistance to fungal diseases and stress factors [2,11,37,56,57,58,59,60,61,62]. This improves planting material quality. Of particular interest is a study of substances of bacterial origin that stimulate the tree species seedlings’ growth and development and protective mechanisms of their influence on plant organisms, which include biologically active metabolites of spore-forming and lactic acid micro-organisms [10,34,35,63,64].
In the present study, we isolated, from natural sources of various ecological niches in the Moscow region and the Republic of Tatarstan, the new strains of micro-organisms. This biological material was used in the development of a complex biological product, stimulating the growth and protection of Scots pine, pedunculate oak and small-leaved linden from fungi diseases caused by Lophodermium pinastri Chev. and Microsphaera alphitoides. The isolates of Bacillus subtilis, Propionibacterium freudenreichii, Lactobacillus plantarum and Streptomyces spp. differ in the ability to produce biologically active metabolites with a pronounced antimicrobial potential against phytopathogenic fungi (Table 1). A number of studies have shown a strain-specific capability of the antagonist bacteria to inhibit phytopathogenic fungi [9,16,65,66,67].
In the course of the study, the isolates were selected for the ability to suppress microscopic fungi, producing hydrolytic enzymes amylase, protease, cellulase and lipase. The most active producers of hydrolases were B. subtilis and L. plantarum. Isolates of Propionibacterium freudenreichii and Lactobacillus plantarum are characterized by their acid-forming activity (Table 2). The antifungal properties of Bacillus subtilis strains are associated with their ability to produce enzymatic complexes (chitinase, chitosanase, protease, cellulase, glucanase, lipase), which effectively break down the main components of a cell membrane of fungi, lipopeptides (surfactin, iturin, fengycin), affecting target phytopathogen cells at a membrane level through interaction with ergosterol [16,66]. This prevents the adhesion of competitive micro-organisms to the parts of a plant organism and induces a plant’s systemic resistance to pathogens and unfavorable abiotic and anthropogenic factors. In addition, the bacterial strains of these species form fungicidal and fungistatic peptides (they are synthesized by the ribosomal multienzyme mechanism), as well as various siderophores (for example, bacilliboctin), which is an action that can be realized through competition for Fe in order to reduce Fe availability for pathogens [68,69,70,71].
The siderophores, produced by Bacillus subtilis, are involved in the suppression of a number of plant diseases [72]. The antifungal potential of bacterial strains of Propionibacterium freudenreichii and Lactobacillus plantarum species is associated with their ability to produce a combination of organic acids (lactic, acetic, propionic, citric, 3-phenyl-lactic, transcinnamic, benzoic and oleamide), fatty acids (methyl esters of 9,12-otadecadienoic and hexadecanoic, 12-hydroxydodecanoic, stearic, lauric and palmitic), bacteriocin-like polypeptides, reuterin, hydrogen peroxide, diketopiperazine and diacetyl [34,63,73,74,75,76,77]. Similar results have been established in the works of many researchers, which show that various Streptomyces spp. produce secondary metabolites with antifungal properties [20,21,22]. The ability of Streptomyces spp. strains to inhibit phytopathogenic fungi is associated with the production of chitinase, betaine, azine, morpholine, pyrazole, fungichromine, actiphenol, ethyl 3-(2-methyl-2-propanyl)-1H-pyrazole-5-carboxylate, 6-amino-5-nitrosopyrimidine-2,4-diol, naphthalene benzaldehyde, carvacrol and phenol, as well as many other compounds.
A feature of the isolates we selected is their ability to grow in a wide temperature range, from 9 to 46 °C, and a pH from 3.0 to 9.0 (Table 2). This opens up the prospects of the joint use of isolates with the herbicidal and fungicidal agents when the acidity of the mixture decreases significantly.
In our study, a Scots pine, pedunculate oak and small-leaved linden seedlings foliar treatment with a Bacterial product LRV increases the plant growth and aboveground and underground biomass compared to the control. The effect of Bacterial product LRV on the seedlings is dose-dependent (Table 4). According to publications [16,62,66], bacteria are able to enhance the growth and development of a host plant, producing biologically active metabolites and inhibiting phytopathogenic micro-organisms. Bacillus spp. strains exert a growth-stimulating activity and increase seed germination energy. These strains also provide plant resistance to stress factors such as frost, drought, high temperatures and fungal and bacterial diseases.
Natural substances formed by bacilli have a number of significant advantages, such as a fairly wide range of biological activity and a positive general effect on plant organism metabolism. Natural substances have a low toxicity and are of high safety for humans and the environment. It is important that these kinds of substances show their activity in an extremely low concentration. This makes their use environmentally and economically beneficial. Some researchers noted [21,78] that streptomycetes and their metabolites are able to provide a stimulating effect on the growth and development of plants. This normalizes plant cell physiology and biochemistry and increases leaf surface index, photosynthesis and respiration intensity, regulating the transpiration coefficient and plant water consumption and reducing the trace elements’ digestible form deficiency. These factors generally affect the productivity and quality of grown products [10,55,79].
In our study, the Biological product LRV at various doses (Table 5 and Table 6) did not significantly affect the powdery mildew and Schutte’s disease in the Scots pine, pedunculate oak and small-leaved linden forest plantations. An increase in the growth and biomass of seedlings’ aboveground and underground parts is associated with isolates selected ability to produce biologically active compounds that can function as plant growth regulators [62,78,80]. An inhibitory effect on other phytopathogenic fungi, different from those presented in this work, capable of infecting and causing diseases of Scots pine, pedunculate oak and small-leaved linden, is highly probable.
The data on the ability of Propionibacterium freudenreichii and Lactobacillus plantarum strains to combine use as a part of a consortium to increase the seedlings’ growth and development have not been available until now. In recent years, interest has increased in research on lactic acid micro-organisms as another class of plant growth promoters and potentially beneficial bacteria against phytopathogens [28,32,42]. There are data on agricultural plants’ increased stress resistance under the influence of an exogenous treatment with metabolites of lactic acid micro-organisms [10], as well as on the successful use of this kind of preparation in medicine, veterinary medicine and viable bacterial cells in crop production [34,35,63,64]. This indicates the possibility of lactobacilli and their metabolites’ wide use in silviculture.
The data on a Bacillus subtilis, Lactobacillus plantarum, Propionibacterium freudenreichii and Streptomyces spp. strains’ combined use as a part of a consortium to increase the productivity of Scots pine, pedunculate oak and small-leaved linden offers the prospect of these valuable tree species’ restoration [16,36,75]. The biologically active preparations that have both growth-stimulating and immuno-inducing effects are of special interest now.
The results obtained during the study indicate a Bacterial product LRV potential to enhance the growth and development of seedlings. Further study of the bacterial product obtained in our research should reveal its ability to increase a tree’s early ontogenesis stage adaptive potential.
The environment plays an important role in silviculture. There are a few possibilities to obtain environmental conditions suitable for the trees. This is a limitation for the new biological preparations, including LRV. A BGT* methodology application will provide a Bacterial product LRV further promotion. The BGT* is a new theoretical, technical and technological basis for long-term soil improvement via intra-soil milling processing [81,82] instead of standard plowing; drought mitigation and freshwater-saving via intra-soil pulse continuously discrete humidification [79,83,84] instead of a standard irrigation and an intra-soil disperse waste ameliorative and nutritional recycling [85,86,87] instead of the standard waste landfills. The BGT* is capable of new, high-level silviculture environmental conditions, providing results that improve up-to-date tree-protective biological preparation applications. A BGT* controlled environment is stable, productive and sustainable compared to standard soil improvement and draught-overcoming methods.

5. Conclusions

From genera Bacillus, Lactobacillus, Lactococcus and Streptomyces isolates, four bacteria strains (Bacillus spp., Propionibacterium spp., Lactobacillus spp., Streptomyces spp.) were selected, and they produced activity against phytopathogenic fungi metabolites that were used for a Biological product LRV creation. A foliar treatment of the Scots pine, pedunculate oak and small-leaved linden seedlings with Biological product LRV increased the growth and biomass of aboveground and underground parts of seedlings compared to the untreated plants. The Biological product LRV provided plant protection from fungal diseases caused by Lophodermium pinastri Chev. and Microsphaera alphitoides.

Author Contributions

Conceptualization, L.R.V., A.R.G. and V.I.E.; data curation, S.N.S., L.L.S. and R.S.M. (Rishat S. Mukhammadiev); formal analysis, S.V.K., S.S.M., M.A.S. and R.S.M. (Rishat S. Mukhammadiev); funding acquisition, M.G.B., R.S.M. (Rinat S. Mukhammadiev) and V.D.R.; investigation, V.V.S., R.K.S., V.I.E., R.S.M. (Rishat S. Mukhammadiev), N.I.B. and I.V.Z.; methodology, L.R.V., V.V.S., R.K.S., N.I.B. and T.M.M.; project administration, R.S.M. (Rinat S. Mukhammadiev) and V.I.E.; resources, A.R.G. and M.A.S.; software, A.R., V.P.K., S.N.S. and S.S.M.; supervision, V.I.C., L.L.S. and V.D.R.; validation, V.V.S., A.R.G., A.R. and T.M.M.; visualization, V.I.C., I.V.Z. and S.V.K.; writing—original draft, L.R.V., R.S.M. (Rinat S. Mukhammadiev), V.P.K. and V.I.C.; writing—review and editing, T.M.M., A.R., V.P.K. and M.G.B. All authors have read and agreed to the published version of the manuscript.

Funding

The work on assessing the antimicrobial properties of micro-organisms isolated from various ecological niches and obtaining compositions based on them was supported by the Russian Science Foundation under grant № 23-26-00161. Description and mathematical processing of the obtained results was carried by the Strategic Academic Leadership Program of the Southern Federal University (“Priority 2030”).

Data Availability Statement

The data presented in this study are available in the figures and tables provided in the manuscript are available upon request.

Acknowledgments

The authors express their deep gratitude to Sh.Z. Validov, Research Laboratory “Microbial Biotechnologies”, IPM&B, Kazan (Volga) Federal University, T.V. Bagaeva, Department of Biochemistry, Biotechnology, and Pharmacology, IPM&B, Kazan (Volga) Federal University for their help and helpful advice in analytical experiments.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Pedunculate oak sowing in 2021: (a) control; (b) double treatment with the Bacterial product LRV in a dose of 10 mL/L.
Figure 1. Pedunculate oak sowing in 2021: (a) control; (b) double treatment with the Bacterial product LRV in a dose of 10 mL/L.
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Figure 2. Small-leaved linden sowing in 2021: (a) control; (b) double treatment with the Bacterial product LRV in a dose of 10 mL/L.
Figure 2. Small-leaved linden sowing in 2021: (a) control; (b) double treatment with the Bacterial product LRV in a dose of 10 mL/L.
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Figure 3. Scots pine sowing 2020: (a) control; (b) Bacterial product LRV 50 mL/L.
Figure 3. Scots pine sowing 2020: (a) control; (b) Bacterial product LRV 50 mL/L.
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Figure 4. Pedunculate oak sowing in 2020: (a) control; (b) Bacterial product LRV 50 mL/L.
Figure 4. Pedunculate oak sowing in 2020: (a) control; (b) Bacterial product LRV 50 mL/L.
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Table 1. Antagonistic activity of selected micro-organism genera against microscopic fungi.
Table 1. Antagonistic activity of selected micro-organism genera against microscopic fungi.
Micro-OrganismTest Culture
Bacillus spp.Propionibacterium spp.Lactobacillus spp.Streptomyces spp.Combination of Four Test Cultures
Fusarium oxysporum+−−−+
Aspergillus flavus+++++
Penicillium spp.+++−+
Candida albicans−+−−+
“−”—absence; “+”—manifestation of antagonistic activity.
Table 2. Properties of selected micro-organisms *.
Table 2. Properties of selected micro-organisms *.
PropertyIsolate
Bacillus subtilisLactobacillus
plantarum		Propionibacterium
freudenreichii		Streptomyces spp.
Morphology of selected micro-organisms
Cell shapesticks, in pairssticks, in pairs, can be chains, depending on the composition of the mediumsticks, in pairs at an angle to each other filamentous form
Gram stain++++
Spore formation+−−−
Mobility−−−+
Cultural properties of selected micro-organisms
The shape of the coloniesroundroundroundrounded convex
Colony colorwhitewhitewhitecream color
Growth of dense nutrient mediauniform growthuniform growth throughout the entire thickness of the medium, near-bottomuniform growth over the entire surface of the medium uniform growth
Physiological and biochemical properties and carbohydrates’ fermentation
Glucose++++/−
Fructose++++
Maltose++−−
Sucrose++−−
Galactose−+++
Lactose+/-+++/−
Arabinose+/−+++
Rhamnose−−++
Sorbitol++/−−+/−
Optimum growth temperature °C35 to 39 33 to 35 30 to 3323 to 25
Optimum pH growth6.9 to 7.2 5.9 to 6.2 6.2 to 7.36.8 to 7.3
Acid-forming activity−++−
Enzymatic activity of selected micro-organisms
cellulolytic++−+/−
proteolytic++++
amylolytic−+−−
lipolytic+/−−+/−+
* “−”—lack of indicator; “+”—availability of indicator.
Table 3. Indicators of 1–3-year-old seedlings of Scots pine, pedunculate oak and small-leaved linden in the Berlibash subdivision of Kaibitsky afforestation enterprise.
Table 3. Indicators of 1–3-year-old seedlings of Scots pine, pedunculate oak and small-leaved linden in the Berlibash subdivision of Kaibitsky afforestation enterprise.
Wood Type and Age of Seedlings Height, cmDiameter, mmGrowth, cmRoot Length, cm
Scots pine 1-year-old5.1 ± 0.1 *0.82 ± 0.02–7.0 ± 0.1
Scots pine 2-year-old 17.7 ± 0.43.84 ± 0.0913.4 ± 0.217.5 ± 0.4
Scots pine 3-year-old25.9 ± 0.54.13 ± 0.1014.6 ± 0.320.1 ± 0.5
Pedunculate oak 1-year-old4.3 ± 0.10.81 ± 0.02–4.9 ± 0.1
Pedunculate oak 2-year-old21.9 ± 0.43.50 ± 0.0815.5 ± 0.319.8 ± 0.4
Pedunculate oak 3-year-old39.5 ± 0.96.9 ± 0.1622.8 ± 0.529.4 ± 0.7
Small-leaved linden 1-year-old4.4 ± 0.10.54 ± 0.01−6.5 ± 0.1
Small-leaved linden 2-year-old12.8 ± 0.33.60 ± 0.0912.3 ± 0.111.4 ± 0.2
Small-leaved linden 3-year-old32.1 ± 0.85.83 ± 0.1414.7 ± 0.319.6 ± 0.3
*—mean error.
Table 4. Biennial seedling biometric indicators in the Berlibash subdivision of Kaibitsky afforestation enterprise.
Table 4. Biennial seedling biometric indicators in the Berlibash subdivision of Kaibitsky afforestation enterprise.
No. Experiment Option *Seedling Planting Density, pcs/m2 (Xav)Seedling Root Length, cm (Xav ± m **)Seedling Height, cm (Xav ± m)Average Weight of Seedling, g;
(%) Relative to Control
Roots (Xav ± m)Aboveground Part (Xav ± m)Total Seedling Weight
(% to Control)
Pedunculate oak
11 3140.1 ± 0.522.7 ± 0.55.2 ± 0.23.4 ± 0.28.6
1 3240.4 ± 0.630.5 ± 0.45.3 ± 0.33.5 ± 0.28.8
223044.5 ± 0.238.0 ± 0.35.7 ± 0.23.7 ± 0.39.4 (109)
2a3150.5 ± 1.140.2 ± 0.46.9 ± 0.74.2 ± 0.411.1 (126)
333447.6 ± 0.439.4 ± 0.25.9 ± 0.33.8 ± 0.29.7 (112)
3a4049.5 ± 0.540.1 ± 0.46.5 ± 0.54.4 ± 0.210.9 (124)
F111.598.8144.1127.8−
Ft 147.9
LSD050.40.90.30.2−
Small-leaved linden
412125.2 ± 0.215.5 ± 0.33.8 ± 0.22.5 ± 0.26.3
1a2026.1 ± 0.415.6 ± 0.43.8 ± 0.42.8 ± 0.36.6
522230.1 ± 0.521.9 ± 0.24.9 ± 0.33.5 ± 0.28.4 (133)
2a2235.8 ± 0.523.7 ± 0.25.6 ± 0.83.9 ± 0.69.5 (144)
632127.3 ± 0.516.5 ± 0.14.0 ± 0.22.9 ± 0.36.9 (109)
3a2326.5 ± 0.316.2 ± 0.23.9 ± 0.42.7 ± 0.36.6 (0)
F31.438.022.837.7−
Ft 38.7
LSD050.20.30.10.2−
Scots pine
71 7315.5 ± 0.621.8 ± 0.52.2 ± 0.21.7 ± 0.63.9
1a 8115.2 ± 0.521.3 ± 0.42.1 ± 0.41.5 ± 0.23.6
827418.3 ± 0.525.1 ± 0.82.9 ± 0.22.8 ± 0.25.7 (146)
2a7719.1 ± 0.226.7 ± 0.43.3 ± 0.23.1 ± 0.46.4 (178)
937818.7 ± 0.422.5 ± 0.43.1 ± 0.21.9 ± 0.35.0 (128)
3a7118.1 ± 0.521.9 ± 0.52.8 ± 0.21.8 ± 0.24.6 (127)
F144.987.465.2139.3−
Ft 155.1
LSD050.60.60.10.2−
* Experiment option: 1. Control—seedlings, which were once sprayed with water; 1a. Control—seedlings, which were sprayed twice with water; 2. Experimental group 1—seedlings treated with the Bacterial product LRV at a dose of 10 mL/L once; 2a. Experimental group 2—seedlings treated with the Bacterial product LRV at a dose of 10 mL/L twice; 3. Experimental group 3—seedlings treated with the Bacterial product LRV at a dose of 4 mL/L once; 3a. Experimental group 4—seedlings treated with the Bacterial product LRV at a dose of 4 mL/L twice. F—calculated Fisher coefficient, Ft—tabular Fisher coefficient). ** m—mean error; LSD05—Least Significant Difference at 5% significance level.
Table 5. Characteristics of Scots pine seedlings (sowing in 2020), 2022.
Table 5. Characteristics of Scots pine seedlings (sowing in 2020), 2022.
Experiment VariantSeedling Root Length, cm (Xav ± m *) Seedling Height, cm (Xav ± m)Schutte’s Disease, %
Control (no processing)22.5 ± 1.69.8 ± 1.288.0
Bacterial product LRV 10 mL/L23.4 ± 2.0 ***10.2 ± 1.3 **89.0
Bacterial product LRV 50 mL/L25.4 ± 2.2 **10.4 ± 1.4 **85.0
Bacterial product LRV 100 mL/L21.3 ± 1.9 **8.5 ± 1.3 *91.0
* m—mean error, ** at p < 0.05, the alternative hypothesis about the presence of differences between groups was accepted; *** at p > 0.05, the null hypothesis of their absence was accepted (post hoc Tukey test).
Table 6. Characteristics of pedunculate oak (sowing in 2020) based on research materials in 2022.
Table 6. Characteristics of pedunculate oak (sowing in 2020) based on research materials in 2022.
Experience VariantSeedling Root Length, cm (Xav ± m *)Seedling Height, cm
(Xav ± m)		Powdery Mildew Susceptibility, %
Control (no processing)42.1 ± 2.012.2 ± 2.975.0
Bacterial product LRV 10 mL/L42.4 ± 2.2 ***11.3 ± 1.6 **79.0
Bacterial product LRV 50 mL/L45.5 ± 1.3 **10.9 ± 1.3 **74.0
Bacterial product LRV 100 mL/L41.9 ± 2.5 ***10.5 ± 1.1 **72.0
* m—mean error, ** at p < 0.05, the alternative hypothesis about the presence of differences between groups was accepted; *** at p > 0.05, the null hypothesis of their absence was accepted (post hoc Tukey test).
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Valiullin, L.R.; Gibadullin, A.R.; Egorov, V.I.; Mukhammadiev, R.S.; Mukhammadiev, R.S.; Sakhnov, V.V.; Singh, R.K.; Sushkova, S.N.; Kozmenko, S.V.; Minkina, T.M.; et al. Ensuring Tree Protection, Growth and Sustainability by Microbial Isolates. Sustainability 2024, 16, 7837. https://doi.org/10.3390/su16177837

AMA Style

Valiullin LR, Gibadullin AR, Egorov VI, Mukhammadiev RS, Mukhammadiev RS, Sakhnov VV, Singh RK, Sushkova SN, Kozmenko SV, Minkina TM, et al. Ensuring Tree Protection, Growth and Sustainability by Microbial Isolates. Sustainability. 2024; 16(17):7837. https://doi.org/10.3390/su16177837

Chicago/Turabian Style

Valiullin, Lenar R., Ascar R. Gibadullin, Vladislav I. Egorov, Rinat S. Mukhammadiev, Rishat S. Mukhammadiev, Vladimir V. Sakhnov, Rupesh Kumar Singh, Svetlana N. Sushkova, Svetlana V. Kozmenko, Tatiana M. Minkina, and et al. 2024. "Ensuring Tree Protection, Growth and Sustainability by Microbial Isolates" Sustainability 16, no. 17: 7837. https://doi.org/10.3390/su16177837

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