1. Introduction
In recent years, due to the increase in maize yields and planting area, the use of chemical fertilizers has risen year by year, exacerbating soil stagnation, acidification, salinization, and reduced use of organic fertilizers, leading to crop yield reductions, increased resistance of phytophagous pests, increased production costs and environmental pollution, and a series of problems [
1,
2].To implement actions and strategies for sustainable food production systems, such as integrated pest management (IPM) and the development of organic agriculture, one of which is based on microorganisms and their metabolites for controlling pest and disease damage to crops, increasing plant "immunity" to abiotic stresses and promoting crop growth [
3,
4,
5,
6].
Metarhizum spp. is widely known as an entomopathogenic fungus, it is widely distributed throughout the world and usually survives in nature as a plant inter-root fungus [
7], a plant endophyte [
8], an insect pathogen [
9], and a soil fungus. In 1883, Sorokin officially named the first species of
Metarhizium as
Metarhizium [
10], and since then the research on
Metarhizium as insect pathogens and biopesticides has begun. Based on previous studies, in addition to increasing plant resistance to pathogenic strains [
11] and having strong insecticidal activity itself, it has been reported in recent years that S. aeruginosa can colonize different plant tissues to promote the growth of the host plant and has a positive impact on the growth indicators of the plant. For example, Enrique González Pérez et al [
12] have shown that M. brunneum can colonize the roots of tomato, maize, Arabidopsis thaliana, peanut, soybean, cotton, coffee, and other plants, and it has a certain promotional effect on the biological indexes such as plant height of the seedlings, root length, and the above-ground and below-ground parts of the plants [
13,
14]. Xinggang Liao et al. showed a significant increase in leaf crown formation, stem length, mean stem leaf, and mean cob biomass in seeds treated with
Metarhizium fungicide as compared to the control, and the main effect on maize yield was during early nutritive growth [
15]. Meanwhile, plant photosynthesis is the basic unit of material accumulation and physiological metabolism in the process of plant growth, and it is also an important means of analyzing the influence of environmental factors on plant growth. It has been shown that endophytic fungi have an extremely strong ability to promote photosynthesis in plants by increasing the supply of carbon dioxide needed for photosynthesis, thus improving plant photosynthesis [
16].
Soil enzymes are mainly derived from the cellular secretions of animals, plants, and microorganisms in the soil and the decomposition of their residues, which are important participants in the soil material cycle and energy transformation, and have a close relationship with the fertility level of the soil, which can characterize the dynamic equilibrium of the soil-microorganism-plant system to a certain extent [
17]. There is an intrinsic link between soil enzymes and plant growth. The application of biotrophic agents can significantly improve the inter-root soil conditions of crops and create a suitable environment for soil enzyme production, and the increase in soil enzyme activity can also promote crop growth and development [
18]. Some studies have shown that fungi fertilizer can regulate the micro-ecological environment of the plant root system and the activity of soil alkaline phosphatase and soil urease, enhance crop resilience, improve the ability of crops to absorb nutrients, and realize the effect of increasing yield and efficiency. However, it has also been shown that there was no significant increase in soil enzyme activities as influenced by environmental factors [
19,
20,
21].
In this project, a strain of M. marquandii was isolated from the understory apomictic material in the Great Xing 'an Range in the preliminary study and was identified morphologically and by 16sRNA and named as M. marquandii SGSF043. Few studies have been reported on the growth promotion of field crops by M. marquandii. In this study, the seeds of "Meiyu" were soaked with the fermentation of M. marquandii. SGSF043.Respectively, to study the effect of this strain on the germination of corn seeds and the growth of seedlings, and at the same time, the inter-root soil enzyme activity of corn seedlings was measured, to clarify the role of M. marquandii. as well as M. marquandii on the promotion of maize and the role of the soil environment of the inter-root zone of seedlings, and the effect of M. marquandii. on the growth and development of maize seedlings was clarified. The results of the study are intended to provide a theoretical basis for further exploring the potential advantages of Metarhizium as a microbial fertilizer for crops.
2. Materials and Methods
2.1. Experimental Strain
M. marquandii SGSF043. Isolated from litterfall in Nanweng River National Nature Reserve in the Great Xing ‘an Range of Heilongjiang province, China. The main plant species are Tilia amurensis Rupr. Quercus mongolica Fisch. exTurcz and Fraxinus mandshurica Rupr. The litter was divided into undecomposed, semi-decomposed, and decomposed layers from top to bottom and the samples were collected in layers. It was brought back to the laboratory and placed in a cool place then dried naturally for isolation and purification. The above strain is kept in the Mycological Collection Center of Wuhan University. (CCTCC No.M2020555).
Source of Experimental Maize: The “Meiyu” sweet and glutinous corn variety, with a harvest period of 75-80 days, was purchased from the seed market and is a common corn variety in Harbin.
2.2. Media Formulation and Fermentation Cultivation
Potato Dextrose Broth Medium (PDB): Weigh 25.0 g of this product in 1L of distilled water, heat, and boil until completely dissolved, pH 8.0, divided, autoclaved at 121℃ for 20 min, ready for use.
Culture conditions for the fermentation broth of M. marquandii SGSF043: The activated M. marquandii SGSF043 was transferred to PDB liquid medium (150 mL/bottle) and placed in a constant temperature shaker at 25 °C, 180 r·min-1 dark condition for 14d. The cultured fungal fermentation broth was filtered through sterile filter paper to obtain the spore-containing fermentation broth, in which the effective amount of viable fungal was measured to be 4.12×109 CFU/mL.
2.3. Experimental Design
The experiment was conducted in August 2022 in the greenhouse of Heilongjiang University. Maize seeds with full were selected to be sterilized by 70% alcohol for 30s and rinsed with sterile water for 5 times. For each sample of 30 maize seeds, before germination, the seeds were soaked in different treatments of fungal solution for 24h, and the soaked seeds were put into 28℃ incubators for germination. A total of five treatments were set up in the experiment: fungus filtrate was diluted with sterile water at 0 (Fermentation Solution); 10; 100; 1,000 times; PDB medium blank control, and three replications of each treatment.
2.4. Effect of Seed Soaking in Fermentation Solution on Germination of Maize Seeds
For each sample of 30 maize seeds, the seeds were immersed in different treatments of Fungal solution for 24 h before germination and subsequently placed in a glass petridish with sterilized filter paper for germination.
The germination was recorded as germination starting from the time when the radicle and endosperm leaked out, and the germination of the maize seeds was observed and recorded daily to calculate the germination potential (%), germination rate (%), and germination index. At the end of the seed germination test the radicle length, radicle thickness, radicle length, radicle thickness, and radicle length of young maize shoots were determined using a straightedge [
22].
In the formula, “GP ”is for germination potential(%); “
A2d ”is the amount of germination seeds in the first two days; “
At ”is the total amount of test seeds.
In the formula, “GE” is germination rate(%); “
Ac” is the amount of germinated seeds when the seed reaches full germination; “
At” is the total amount of test seeds.
GI is the germination index; “Gt“ is the amount of germination on day t; “Dt ”is the corresponding amount of days to germination.
2.5. Determination of Biomass in Maize Seedlings
Seeds were treated with fungus solution before sowing in field plot trials, and samples were taken 20 d after corn sowing, five seedlings were selected for each treatment, and the roots of the plants were cleaned with tap water and the surface water was blotted out with filter paper to measure the plant height, root length, ground stem thickness, aboveground fresh weight, and belowground fresh weight.
2.6. Determination of Photosynthetic Indexes and Chlorophyll SPAD content in Maize Seedlings
Photosynthetic gas exchange parameters were determined using a LI-6400 photosynthesizer (USA). On September 2, 2022, from 9 a.m. to 11 a.m., plants with consistent growth were selected in each treatment to determine the relevant indexes, and each treatment was measured three times. The measured indices included net photosynthetic rate (A), transpiration rate (E), intercellular CO2 concentration (Ci), and stomatal conductance (GH2O).
The SPAD values of maize leaves were measured using a chlorophyll meter (FK-YL01) in the middle of the maize functional leaves, and each treatment was measured six times.
2.7. Determination of Inter-root Soil Enzyme Activities in Maize Seedlings
Soil Alkaline Phosphatase Kit (Solarbio LIFE SCIENCES, BC0285), Soil Neutral Protease Kit (Solarbio LIFE SCIENCES, BC0275), and Soil Uidase kit (Solarbio LIFE SCIENCES, BC0125) were used to determine the Enzyme activity in rhizosphere soil of maize seedlings in according to the manufacturer's instructions.
2.8. Statistical Analysis
Microsoft Excel and SPSS 27 were used for data processing and statistical analysis and GraphPad Prism 9 software package was used for plotting. The significance of differences between groups was tested at the 0.05 level by the LSD test in one-way ANOVA (one-way analysis of variance).
3. Results
3.1. Effect of Fermentation Solution Soaking on Germination of Maize Seeds
The data in
Table 1 and
Table 2 show that the fermentation broth (Filtrate Stock,0) treatment group had some inhibitory effect on seed germination.
Table 1 Among all the dilution treatments, the highest seed germination rate of 97% was recorded in the group treated with 10 times dilution of solution and was significantly higher than the other treatment groups with a germination time of 1 d (P<0.05). The differences in germination potential, germination index, and Control (PDB liquid medium) between the 10 times dilution and 1000 times dilution treatment groups were not significant (P>0.05) and were higher than the 100 times dilution treatment group.
As can be seen from the group diagram of some samples 2d after soaking in
Figure 1, among all the dilution multiples, the embryo buds and embryonic axes of seeds in the treatment groups of 100 times fermentation solution and 1,000 times fermentation solution had the best growth, and with the increase of the dilution multiples of the fungal solution, the number of white lateral roots around the main roots of the seeds increased gradually in the form of a regular radial shape. Combined with the data in
Table 2, it can be seen that the radicle thickness, radicle length, endosperm thickness, and embryonic axis length of maize seeds in the treatment group of 1,000 times dilution increased compared to the other treatment groups. They increased by 13.8%, 32.5%, 10.43%, and 27.1%, compared with the control group respectively. Among them, the greatest effect was on the germ length and embryonic axis length of maize seeds.
3.2. Effect of Maize Seedling Biomass
A box-and-line plot was chosen to statistically graph the degree of data dispersion for each indicator of maize seedling biomass.
Figure 2 shows the effect of
M. marquandii on the biomass of maize seedlings, indicating that different dilutions of the fermentation filtrate of the test strain can promote the growth of maize seedlings, and the indicators of plant height, root length, diameter and thickness, aboveground fresh weight and underground fresh weight are higher than those of the control treatment. Different dilution concentrations had different effects on the seedling morphology of maize plants.
In
Figure 2 (a), it can be seen that the difference in plant height between the 10 times dilution treatment group and the control group was significant (P<0.05) and also slightly higher than the 100 times and 1,000 times treatment groups. The relatively large dispersion of the 1,000 times plant height data indicates that the data fluctuated within the group.
The median of 10 times dilution was 58.5 cm, 100 times dilution was 52.9 cm, 1,000 times dilution was 53.4 cm, and the median of the control group was 48.4 cm. The height of maize seedlings in each fungal dilution treatment group was higher than that of the control treatment group, which was increased by 20.90%, 9.30%, and 10.33%, compared with the control group, respectively.
In
Figure 2 (b), it can be seen that the root length of plants in all dilution treatment groups differed significantly (P<0.05) from that of the control group. 100 times solution root length data were more discrete, suggesting that the data fluctuated within the group. The median of 10 times dilution was 17.3 cm, the median of 100 times dilution was 14.7 cm, the median of 1,000 times dilution was 16.6 cm, and the median of the control group was 10.2 cm. The height of maize seedlings in each bacterial dilution group was higher than that of the control group, which was increased by 69.61%, 30.61%, and 62.75% compared with that of the control group, respectively.
In
Figure 2(c), it can be seen that the difference between the above-ground fresh weight of the 100 times dilution treatment group and the other treatment groups was significant (P<0.05). The median of 10 times dilution was 8.425 g, 100 times dilution was 12.04 g, 1000 times dilution was 8.791 g, and the median of control treatment was 5.356 g. The above-ground fresh weight of maize in each dilution treatment group was increased by 57.3%, 125.8%, and 64.13%, compared with the control group, respectively.
In
Figure 2 (d), the difference between the underground fresh weight of 100 times dilution treatment group and 1,000 times dilution treatment group was not significant (P>0.05). The median of 10 times dilution was 0.742 g, 100 times dilution was 1.038 g, 1000 times dilution was 0.943 g, and the median of control treatment was 0.457 g. The above-ground fresh weight of maize in the treatment groups of each bacterial dilution was increased by 62.36%, 127.13%, and 106.35%, compared with the control group, respectively.
From
Figure 2(e), it can be seen that the maize seedling ground diameter thickness of each dilution treatment group was higher than that of the control treatment group, and the data were stable within the group. The median of 10 times dilution was 8.76 mm, 100 times dilution was 7.89 mm, 1,000 times dilution was 8.2 mm, and the median of the control treatment group was 6.6mm, and the treatments of each dilution increased the diameter of the ground roughness by 32.73%, 19.55%, and 24.24%, compared with the control group, respectively.
Figure 2.
Effect of Maize Seedling Biomass. (a) Plant height; (b) Root length; (c) Shoot Fresh Weight; (d) Root Fresh Weight; (e) Ground diameter. The results are presented as the mean±standard deviation of three replicates. Different letters indicate the difference between different treatment groups (P<0.05).
Figure 2.
Effect of Maize Seedling Biomass. (a) Plant height; (b) Root length; (c) Shoot Fresh Weight; (d) Root Fresh Weight; (e) Ground diameter. The results are presented as the mean±standard deviation of three replicates. Different letters indicate the difference between different treatment groups (P<0.05).
3.3. Effect of Photosynthetic Indexes and Chlorophyll SPAD in Maize Seedlings
Different dilution treatments of the fermentation filtrate of M. marquandii had an effect on the photosynthetic properties of maize seedling leaves. Among them, different dilutions of fermentation solution increased the net photosynthetic rate (A) of maize seedlings (Figure a), which differed significantly (P<0.05) compared with the treatment of soaking in PDB sterilized liquid medium, and the data within the group of 10 times dilution of the fermentation solution were stable compared with those of the 100 times dilution treatment group and the 1,000 times dilution treatment group.
The transpiration rate (E) (Figure b) and stomatal conductance (GH2O) (Figure d) of the plants in the 100 times dilution treatment group differed significantly (P<0.05) from those of the control treatment group. The transpiration rate of plants corresponding to 10 times dilution, 100 times dilution, and 1,000 times dilution was the most significant at 3.04 mmol/(m2·s), 3.48 mmol/( m2·s), and 2.5 mmol/( m2·s ), compared to 1.66 mmol/( m2·s) in the control treatment group, respectively; The highest leaf stomatal conductance of 132.6 mmol/( m2·s) was recorded for the 100 times dilution.
The control treatment group plants had the highest interstitial carbon dioxide concentration (Ci) of 121 µmol/( m2·s), and the 1,000 times dilution corresponded to the lowest interstitial carbon dioxide concentration (Ci) of 57.4 µmol/( m2·s). The interstitial carbon dioxide concentrations (Ci) of the plants in the 10 times dilution and the 100 times dilution were 73.2 µmol/( m2·s), and 85.4 µmol/( m2·s), respectively.
The highest chlorophyll SPAD value of 41.12 was recorded for maize seedlings in the 100 times treatment group; 40.94 for the 10 times treatment group; 38.62 for the 1,000 times treatment group; and 38.92 for the control treatment group.
Figure 3.
Effect of Maize Seedling Biomass. (a) A;(b) E;(c) Ci;(d) GH2O;(e) SPAD. The results are presented as the mean±standard deviation of three replicates. Different letters indicate the difference between different treatment groups (P<0.05).
Figure 3.
Effect of Maize Seedling Biomass. (a) A;(b) E;(c) Ci;(d) GH2O;(e) SPAD. The results are presented as the mean±standard deviation of three replicates. Different letters indicate the difference between different treatment groups (P<0.05).
3.4. Inter-root Soil Enzyme Activities in Maize Seedlings
Soil alkaline phosphatase (
Figure 4a), soil neutral protease (
Figure 4b), and soil urease (
Figure 4c) were measured in the inter-root of maize seedlings in different treatment groups. Differences between treatments in soil alkaline phosphatase and soil urease were not significant (P>0.05). Soil neutral protease (
Figure 4b) was significantly (P<0.05) higher in the 100 times dilution neutral protease than in the other three treatment groups. The difference between 10 times dilution and 1,000 times dilution was not significant (P>0.05) and the values were lower than the control treatment group.
4. Discussion
It has been shown that the association between
Metarhizium spp. and plants and insects is mainly based on the nutritional basis [
23].
Metarhizium spp. can transfer nitrogen produced by insects to plants for plant growth by parasitizing and killing the insects [
8]. Plants provide carbon for
Metarhizium spp. Through photosynthesis [
24], and vigorously growing plants can provide a more stable habitat for
Metarhizium spp. , while the parasitic nature of
Metarhizium spp. can further protect the healthy growth of plants.
Marquandomyces Marquandii belongs to the genus of non-core
Metarhizium spp. which are widely present in soil.
Marquandomyces Marquandii belongs to the genus of non-core
Metarhizium spp. which are widely present in soil. Some studies have shown that the secondary metabolites of
M. marquandii not only have antimicrobial and antitumor activities [
25], but also have the potential to be used in the removal of environmental pollutants (heavy metal pollution and pesticide residues, etc.), the control of crop pests and diseases, and the promotion of plant growth [
26,
27]. However, only a few studies have been reported on the plant growth promotion of this strain.
The experimental study was undertaken using the pre-existing Isolation and identification of
M. marquandii SGSF043 from the Litter under the forest. Diluting the fungal fermentation broth at different multiples in the growth stabilization period and soaking the seeds, it was found that the seeds of undiluted fungal fermentation broth did not germinate, which indicated that the fermentation broth of
M.marquandii SGSF043 had an inhibitory effect on the germination of maize seeds. The fermentation solution diluted by sterile water began to show the promotion effect on seed germination, among which, the highest seed germination rate was found in the group treated with 10 times dilution of bacterial solution, and the germination time was 1 d. After the seed germination test was completed, we continued to observe the growth of the seed germ and found that the germ and embryonic axis of seeds in the groups treated with 100 times fermentation solution and 1,000 times fermentation solution were the best in growth, and the seeds with 1,000 times dilution of seed had the most root hairs on the lateral root, which The seeds of 1,000 times dilution had the most lateral root hairs, which were in regular radial shape. Moreover, many studies have demonstrated that exogenous microorganisms can influence the root system configuration through colonization of the inter-root zone, mainly by affecting the growth of primary roots, lateral roots, and root hairs [
28]. This is basically consistent with the results of the present study.
During the seedling growth period, different dilutions of the fermentation broth of
Marquandomyces marquandii can promote the increase of various indicators of maize seedling height, root length, ground diameter, aboveground and underground fresh weight, and have different effects on the root morphology of maize seedlings. The growth indicators of the bacterial fermentation broth treatment group were better than those of the control group. In general, when the seeds soaked in the fermentation broth were planted into the soil, the 100 times dilution was the best for plant growth and development. The growth promotion of plants is first manifested in the changes of roots. Roots are not only an absorption organ, but also an organ that senses environmental changes. The growth and development of plant roots are closely related to the environment [
29]. After the ermentation broth treatment, the root length of the seedlings is longer than that of the control group. The fresh weight of corn stems and roots in 100 times dilution solution is significantly higher than that of other treatment groups. With the change of environment, the seeds after the same treatment are cultured in Petri dishes and soil respectively, As the environment becomes more complex, the impact of
Marquandomyces marquandii on plants has also changed accordingly. For example, the 1,000 times dilution in the Petri dish promotes the growth of seed germ and radicle. The best growth and development index of seedlings in the soil is 100 times dilution, and the required concentration of
Marquandomyces marquandii increases. Therefore, it is presumed that
Marquandomyces marquandii SGSF-043 may achieve the growth-promoting effect on maize by regulating the expression of genes related to lateral root formation, auxin synthesis, and transportation in maize, which needs further research through molecular research methods.
In this experiment, we found that the leaf
A (Net Photosynthetic Rate),
E (Transpiration Rate), and
GH2O (Stomatal Conductance) of each treatment group of the fermentation solution were higher than those of the treatment group, and the maize soaked by
M. marquandii SGSF-043 could improve the photosynthetic capacity by increasing the photosynthetic gas exchange capacity and water uptake, and this is also consistent with the findings of the previous study mentioned that when the host endophytic fungi infested the plant roots, the fungus could consume the photosynthesized products in the host body to make the photosynthesis increase [
30]. The interesting finding is that the
Ci (Intercellular carbon Dioxide Concentration) of each treatment group of the fermentation solution was lower than that of the blank control treatment group, which is hypothesized in conjunction with the results of the experiments corresponding to the other photosynthetic indexes, probably when photosynthesis is accelerated, the plant absorbs more carbon dioxide for photosynthesis from the environment and from other cells, which makes the intracellular inter-cellular carbon dioxide concentration lower, and the process of the plant absorbing carbon dioxide and water and converting them into chemical energy (glucose and oxygen) under the action of light. On the other hand, the cause of the decrease in intercellular CO
2 concentration could also be the accelerated rate of CO
2 consumption by the plant, resulting in a slower rate of CO
2 production. If the respiration of plants, plant chlorophyll content is one of the main indicators of the photosynthetic capacity of plants [
31]. In the results of this study, the chlorophyll content of the plants in the treatment groups of fermentation solution did not differ much from the chlorophyll content of the control group, which is slightly different from the results of previous studies.
The addition of beneficial microorganisms can directly or indirectly with the microorganisms in the soil around the roots to varying degrees to increase soil enzyme activity, while closely related to the organic matter content and so on. Soil enzyme activities can reflect the type and strength of biochemical reactions in soil [
32]. In this experiment, the inter-root soil alkaline phosphatase and soil urease contents of the fermentation solution treatment group were slightly higher than those of the control group. Soil-neutral protease content was significantly higher in the fermentation solution diluted 100 times in the treatment than in the other treatment groups. Inorganic nitrogen is released through the mineralization of organic nitrogen, which is an important source of nitrogen for plant growth. Soil inorganic nitrogen content characterizes the soil's ability to supply nitrogen. It has been shown that the highest neutral protease activity was found in soil with ammonium nitrogen alone, while the neutral protease activity was enhanced with the increase of ammonium nitrogen percentage [
33]. It can be hypothesized that
M. marquandii may produce inorganic ammonium nitrogen or recruit beneficial microorganisms that can produce ammonium nitrogen, thus directly or indirectly promoting the uptake and utilization of nutrients in the soil by plants, and the mechanisms related to the plant-promoting potential of
M.marquandii will be investigated in more detail in the future.
5. Conclusions
The fermentation broth of Marquandomyces marquandii SGSF-043 can promote the germination of maize seeds and increase the biomass of corn seedlings at a suitable concentration. In the maize seed germination tests, the seed germination effect treated with 10 times dilution of bacteria fermentation broth was the best one. After the seed germination test, as the seed germs continued to grow, the radicle length, radicle diameter, germ length, germ diameter, and hypocotyl length of the seed were measured. It was found that the 100 times dilution and 1000 times dilution treatment groups had the best effect. Among them, the 1,000 times dilution had the most obvious effect on the elongation and growth of lateral roots around the seed main root, which increased by 13.8%, 32.5%, 10.43%, and 27.1% respectively compared with the control group. According to the field plot test, there were no significant differences in chlorophyll content among these treatments. According to the measurement data of maize growth and development, photosynthesis, and rhizosphere soil-related enzyme activities, it can be determined that the best effect of promoting corn growth and development is when the fermentation broth of Marquandomyces marquandii is diluted 100 times. It shows that Marquandomyces marquandii has the potential for development and utilization, which can be further studied and applied.