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Article

Trichoderma Rhizosphere Soil Improvement: Regulation of Nitrogen Fertilizer in Saline–Alkali Soil in Semi-Arid Region and Its Effect on the Microbial Community Structure of Maize Roots

by
Yicong Li
1,
Jianming Cui
1,
Jiarui Kang
1,
Wei Zhao
1,
Kejun Yang
2 and
Jian Fu
1,2,*
1
Agricultural College, Heilongjiang Bayi Agricultural University, Daqing 163319, China
2
Heilongjiang Key Laboratory of Modern Agricultural Cultivation Technology and Crop Germplasm Improvement, Daqing 163319, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(10), 2340; https://doi.org/10.3390/agronomy14102340
Submission received: 14 September 2024 / Revised: 5 October 2024 / Accepted: 8 October 2024 / Published: 11 October 2024
(This article belongs to the Section Soil and Plant Nutrition)
Browse Figures
Versions Notes

Abstract

:
In order to reduce the actual impact of a saline–alkali environment on maize production in semi-arid areas, it is particularly important to use the combined fertilization strategy of Trichoderma microbial fertilizer and nitrogen fertilizer. The purpose of this study was to investigate the effects of different concentrations of nitrogen fertilizer combined with Trichoderma on improving the structural characteristics and ecological functions of maize rhizosphere microbial community in semi-arid saline–alkali soil. Through the microbiome analysis of maize rhizosphere soil samples with 60 kg N·ha−1 (N1) and 300 kg N·ha−1 (N2) nitrogen fertilizer combined with Trichoderma (T1) and without Trichoderma (T0), we found that the combination of Trichoderma and different concentrations of nitrogen fertilizer significantly affected the structure of bacterial and fungal communities. The results of this study showed that the combination of Trichoderma and low-concentration nitrogen fertilizer (N1T1) could improve soil nutritional status and enhance its productivity potential, revealing the relationship between beneficial and harmful fungal genera, microbial diversity and abundance, and crop biomass, which is of great significance for improving agricultural production efficiency and sustainable development.

1. Introduction

Maize is a key crop for global food security, and its yield occupies a place in the total grain yield of the world. Northeast China is known as one of the world’s three ‘golden corn belts’, and its corn production accounts for 40% of the country’s total output. However, at the same time, the Plain, as one of the three major areas of soda saline–alkali land in the world, the area of saline–alkali land is increasing. Land salinization is becoming a worldwide environmental problem that seriously affects soil quality. The main reason for the low productivity of saline–alkali soil is poor microbial activity [1]. Therefore, in order to ensure food security, Trichoderma, as a biological control agent with broad application prospects [2], enhances the growth quality and stress resistance of plants through interaction with plants. In addition, Trichoderma has also shown positive effects in its role as a biological fertilizer and in improving crop resistance to biotic and abiotic stresses [3]. They inhibit the growth of pathogens by establishing beneficial microbial communities, i.e., microbial aggregates, while promoting the healthy growth of plants, improving nutrient uptake efficiency, and enhancing plant host resistance [4,5,6,7]. The application of Trichoderma can restore healthy and active soil microbial flora in a relatively short period of time, effectively improve soil properties, improve soil fertility, and thus play a positive role in plant growth. These benefits are closely related to the effect of metabolites produced by Trichoderma in the interaction with plants and other microorganisms [8].
The application of Trichoderma not only helps to improve crop yield and quality but also reduces the dependence on chemical fertilizers [9], thereby promoting the sustainable development of agriculture. As a key nutrient element for plant growth, nitrogen (N) has a significant impact on increasing crop yield [10,11] and is one of the most important limiting factors in agricultural production [12]. It is generally believed that increasing the application of nitrogen fertilizer can bring rich output. However, the excessive use of nitrogen fertilizer not only fails to further increase crop yield but also may lead to a series of environmental problems [13,14], such as nitrogen loss and greenhouse gas emissions, which pose a threat to the health of ecosystems. In actual agricultural production, farmers tend to apply higher doses of nitrogen fertilizer in order to pursue higher crop yields [15,16]. Therefore, it has become an important challenge in agricultural research to find the optimal balance point of the combined application of microbial agents and nitrogen fertilizer to achieve the sustainability of crop production. Although Trichoderma has a certain promoting effect in ordinary agricultural soil, the promoting effect of Trichoderma combined with nitrogen fertilizer in saline–alkali soil in semi-arid areas is rarely reported.
The purpose of this study was to explore the effects of Trichoderma combined with nitrogen fertilizer on the characteristics of maize rhizosphere microbial community in Northeast China and to evaluate the potential effects of Trichoderma combined with nitrogen fertilizer on soil microbial richness, diversity, and ecological function. At the same time, we will also analyze the bacterial and fungal community structure to determine the main phylum/genus in each rhizosphere soil system. The core hypothesis of this study is that in the semi-arid saline–alkali soil, the combined application of Trichoderma and nitrogen fertilizer will create a more favorable microbial ecological environment by promoting the improvement of soil microbial diversity and richness, thus effectively improving soil fertility and promoting crop growth and nutrient absorption under saline–alkali conditions. We will provide theoretical support for the sustainable development of agriculture through an in-depth analysis of the rhizosphere microbial community under the combined use of Trichoderma and nitrogen fertilizer.

2. Materials and Methods

2.1. Test Sites and Varieties

The experiment was carried out in the experimental station of Heilongjiang Bayi Agricultural University in Daqing City, Heilongjiang Province, China (46°37′ N, 125°11′ E) in 2020. The extreme minimum temperature in the test area is −39.2 °C, the average temperature of the hottest month is 23.3 °C, the extreme maximum temperature is 39.8 °C, the average annual frost-free period is 143 days, the annual precipitation is 427.5 mm, and the annual evaporation is 1635 mm. The farmland soil type is meadow saline–alkali soil. The basic physical and chemical properties of soil are as follows: total nitrogen 1.07 g/kg, alkali-hydrolyzable nitrogen 123.78 mg/kg, available phosphorus 46.76 mg/kg, available potassium 189.36 mg/kg, organic matter 21.39 g/kg, pH 8.41, HCO3− 0.142 g/kg, Cl− 0.082 g/kg, sodium adsorption ratio 0.374, and soluble salt content 1.24 g/kg. The tested maize variety was Xianyu 335.

2.2. Experimental Design

The experiment was sown on 15 May 2020. The planting area was divided by random block design. Each treatment group was allocated an area of 42 m2, with a total of 6 ridges (0.7 m × 10 m). Maize seeds were sown at a density of 82,500 plants/hectare with a row spacing of 0.7 m.
The treatment method was designed with two factors: different nitrogen application levels and Trichoderma. The nitrogen application levels were 60 kg N ha−1 (N1) and 300 kg N ha−1 (N2), and Trichoderma was set as no Trichoderma agent (T0) and Trichoderma agent (T1). There were a total of four treatments. A blank control group was set (Table 1). Trichoderma spores (GenBank accession number: KJ541741) were first activated in PDA medium at 185 r/min and 28 ± 2 °C and then prepared into spore suspension (1 × 109 CFU/mL), which was inoculated into sterilized solid matrix (1:20, v/w) and incubated at 28 °C for 10 days. The application amount of Trichoderma agent was 1.4 g conidia powder mixed with 200 mL water and inoculated into the root soil in the form of solution on the 25th and 45th days after the emergence of maize seedlings. The same amount of water was poured at the same time without Trichoderma treatment. The nitrogen fertilizer used in the experiment was ammonium sulfate, and the application amount of phosphorus and potassium fertilizer was the same in each treatment. During the experiment, we followed the conventional agronomic measures in the semi-arid region of Northeast China to ensure the authenticity and reliability of the experimental results.

2.3. Determination of Plant Nutrient Elements

The mature stage of maize was sampled, and the plant was divided into stem, leaf, and grain organs. The samples of each organ were killed at 105 °C for 30 min in the oven, dried to constant weight at 70 °C, and ground. Total nitrogen, phosphorus, and potassium were determined by concentrated sulfuric acid–hydrogen peroxide digestion. The nitrogen content was determined by the Kjeldahl method, the phosphorus content was determined by the vanadium molybdenum yellow colorimetric method, and the potassium content was determined by atomic absorption spectrophotometry.

2.4. Rhizosphere Soil Sampling

In 2020, at the maturity stage of maize, each treatment was sampled at 5 points, and the maize plants with soil were dug out. The larger granular soil on the roots of the plants was gently shaken off, and the rhizosphere soil adhered to the fibrous roots was collected by a fine brush into a sterile self-sealing plastic bag. The soil samples were stored in a refrigerator at −80 °C and analyzed within 1 day.

2.5. DNA Extraction

Rhizosphere soil microbial diversity was measured at the maturity stage. Microbial DNA was extracted from each soil sample according to the instructions on the E.Z.N.A.® soil DNA kit (OMEGA Bio-Tek, Norcross, GA, USA). The variable region V3–V4 of the bacterial 16S rRNA gene was amplified by PCR with 338F/806R. The ITS1-ITS2 of the fungal ITS rRNA gene was amplified by primers ITS1F/ITS2R. The PCR reaction system was as follows: 4 μL 5× FastPfu Buffer, 2 μL dNTPs (2.5 mM), 0.8 μL pre-primer (5 μM), 0.8 μL reverse primer (5 μM), 0.4 μL FastPfu Polymerase, 0.2 μL BSA, 10 ng DNA template, and 20 μL water. After pre-denaturation at 95 °C for 3 min, 27 bacterial or 35 fungal amplifications were performed at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, respectively. Finally, the bacteria were stably extended at 72 °C for 10 min and then stored at 10 °C. Purified amplicons were assembled at an equal molar concentration and sequenced on the Illumina MiSeq PE 250 platform at 2 × 300.

2.6. Statistical Analysis

Microsoft Excel was used to organize the collected data. Data tables were created using SPSS 27.0 software and Microsoft Office Excel 2010.
R software 4.4.1 was used for common and endemic species statistics, community composition analysis, and species abundance cluster analysis. The representative sequences of the top 15 OTUs (Operational Taxonomic Units) in the overall relative abundance were selected, and the FastTree software 2.1 was used to construct the tree by infinite comparison. At the same time, the relative abundance of each OTU and the species annotation confidence information of the representative sequence were combined, and the ggtree software 3.13.1 package was used to visualize the system evolution. Based on the OTU abundance table, four alpha diversity indices (Simpson, chao1, Shannon, and ACE) were performed using usearch-alpha_div (V10, http://www.drive5.com/usearch/ (accessed on 23 April 2024)) and R packages. Based on the OTU abundance table, PCoA analysis was performed using the vegan package of R software with the bray_curtis distance algorithm. The Linear Discriminant Analysis Effect Size (LEfSe) was used to find the microbial groups that were significantly affected by each treatment, and the LDA histogram was drawn using groups with LDA scores > 3. FAPROTAX and FUNGuild were used to predict bacterial and fungal functions. The structural equation model (SEM) is widely used in the field of mathematical statistics. It can be used to test the complex path relationship network and establish the structural equation model by AMOS 24 software. The graphical abstract was drawn by Figdraw.

3. Results

3.1. Changes of α Diversity in Rhizosphere Soil under Different Treatments

According to Figure S1, in the bacterial community, compared with T0, the Simpson index under T1 conditions, N1 and N2 treatments increased by 8.66% and 13.17%, respectively, and the Shannon index decreased by 6.18% and 7.23%, respectively. The ACE index showed that N2T1 was 0.35% higher than N2T0.
In the fungal community, compared with T0, the Simpson index decreased by 4.89% and 17.92% under N1 and N2 treatments, respectively, and the Shannon index increased by 10.29% and 10.23%, respectively. The ACE index under T1 conditions, N1 and N2 treatments were 3.22% and 22.60% higher, respectively.

3.2. Bacterial and Fungal Community Composition and Cluster Analysis of Rhizosphere Soil under Different Treatments

The dominant bacterial communities (relative abundance > 1%) at the phylum level (Figure 1a) were mainly Proteobacteria (28.73–34.42%), Acidobacteria (15.20–22.81%), Actinobacteria (9.99–17.68%), Chloroflexi (7.14–8.57%), Planctomycetes (7.04–8.45%), Bacteroidetes (4.98–7.82%), Gemmatimonadetes (5.70–7.53%), and so on. Clusters were formed in N2 and N1 treatments, respectively. There was no significant difference in the composition and structure of the dominant phylum among the treatments, but there were significant differences in the abundance of the dominant phylum, especially the application of Trichoderma treatment. Compared with T0, under T1 conditions, the abundance of Proteobacteria and Bacteroidetes in N1 and N2 treatments decreased by 4.84%, 6.09%, and 18.63%, 29.49%, respectively. The abundance of Acidobacteria and Chloroflexi increased by 10.64%, 12.02% and 9.20%, 19.60%, respectively. At the genus taxonomic level (Figure 1b), the dominant genera (relative abundance > 1%) of the bacterial community were mainly MND1 (7.35–13.40%), RB41 (6.09–12.76%), Haliangium (3.08–6.49%), Ellin6067 (4.04–4.55%), Nocardioides (2.98–5.85%), Lysobacte (2.05–5.64%), Sphingomonas (2.15–5.79%), Pirellula (3.06–3.74%), Gaiella (2.30–4.57%), Bryobacter (2.64–3.47%), and Subgroup 10 (2.09–3.20%). The clustering situation is similar to the phenomenon observed at the gate level. Compared with T0, the relative abundance of MND1, RB41, Haliangium, Ellin6067, and Gaiella under N1 treatment increased by 33.82%, 17.09%, 11.39%, 7.41%, and 19.69% under T1 condition. The relative abundance of RB41, Ellin6067, Nocardioides, Gaiella, Pirellula, and Subgroup 10 increased by 39.46%, 6.04%, 43.28%, 36.06%, 5.18%, and 53.38% under N2 treatment.
At the phylum level (Figure 1c), the dominant fungal communities (relative abundance > 1%) were mainly Ascomycota (45.80–62.72%), Basidiomycota (11.42–24.78%), Mortierellomycota (1.23–2.91%), and Cercozoa (1.20–2.72%). Compared with T0, under T1 conditions, the Ascomycota decreased by 15.62% and 6.66% under N1 and N2 treatments, respectively. The abundance of Basidiomycota increased by 38.06% and 67.09%, respectively. The composition (relative abundance > 1%) of the dominant fungal communities at the genus level (Figure 1d) was Ophiosphaerella (1.27–9.30%) and Mortierella (1.17–3.17%). Compared with T0, the relative abundance of Conocybe, Mortierella, Kernia, Penicillium, and Acremonium under N1 treatment increased by 95.80%, 155.17%, 2028.32%, 136.18%, and 110.90% under T1 condition. The relative abundance of Mortierella, Penicillium, Acremonium, and Podospora increased by 53.75%, 139.51%, 69.49%, and 54.72% under N2 treatment. Through further analysis of the composition of fungal genera (Figure 1e), it was found that under T1 conditions, the relative abundance of beneficial bacteria Mortierella, Penicillium, and Acremonium under N1 and N2 treatments increased by 162.18% and 43.89%, 142.11% and 122.78%, and 117.44% and 58.57%, respectively. Trichoderma treatment significantly increased the abundance of beneficial bacteria, and the effect was better under N1 conditions. In the analysis of harmful bacteria (Figure 1f), the relative abundance of Ophiosphaerella and Gaeumannomyces decreased by 35.57% and 14.68%, 19.27% and 34.78% under N1 and N2 treatments under T1 conditions. Trichoderma koningii inhibited the abundance of harmful fungi, especially under high nitrogen conditions. The inhibitory effect on some harmful fungi was more significant.

3.3. β Diversity of Microbial Community in Different Treatments

Principal coordinate analysis (PCoA) was performed based on the Bray–Curtis distance (Figure S5a). The results showed that N1 treatment and N2 treatment were well separated. At the same time, the coverage of T1 treatment clustering is greater, and there is a significant difference with the T0 treatment. The PCoA map showed a clear clustering of rhizosphere bacterial communities. The first axis (54.8%) and the second axis (27.3%) explained 82.1% of the total variation of the bacterial community. Further analysis at the genus level (Figure S5b) showed that the community composition under N1T0 and N1T1 treatments was similar but significantly separated from N2 treatment. The PCoA map showed a clear clustering of rhizosphere bacterial communities, with the first axis (45.4%) and the second axis (14.4%) explaining 59.8% of the total variation of bacterial communities.
PCoA analysis of fungal community structure (Figure S6a) showed that at the phylum level, the clustering of rhizosphere fungal communities under T1 treatment was significantly separated from that under T0 treatment. The PCoA diagram showed a clear clustering of the rhizosphere fungal community. The first axis (51.5%) and the second axis (27.1%) explained 78.6% of the total variation of the fungal community. In addition, the PCoA analysis of the fungal community structure at the genus level (Figure S6b) showed that the rhizosphere fungal community under the T0 treatment was obviously clustered and significantly separated from the T0 treatment. The PCoA diagram showed a clear clustering of the rhizosphere fungal community. The first axis (49.1%) and the second axis (18.3%) explained 67.4% of the total variation of the fungal community.

3.4. LEfSe Species Difference Analysis

In terms of the bacterial community, LEfSe analysis revealed 56 bacterial genera (Figure S7), and 15,14,12,8 and 7 biomarkers were identified in Con, N1T0, N1T1, N2T0, and N2T1, respectively. The LDA values of the main discriminant groups Phycisphaerae, Tepidisphaerales, and D2101 soil group in N1T0 treatment were all greater than 4. The LDA values of 8 different levels in Proteobacteria were greater than 3 in N1T0 treatment, including the main discriminant groups of Alphaproteobacteria and its Sphingomonadales, Sphingomonadaceae and Novosphingobium. Xanthomonadales and uncultured bacterium at the species level of Xanthomonadaceae, Lysobacter and Rhodanobacteraceae. Similarly, the main discriminant groups in N1T1, Blastocatellia Subgroup 4 in Acidobacteria, Pyrinomonadales, Pyrinomonadaceae, and RB41 species had LDA values greater than 4, which could be used as specific biomarkers. The LDA values of the main discriminant groups in N2T1, Thermoleophilia, Solirubrobacterales, Solirubrobacteraceae, Solirubrobacter, Blastococcus, and Blastococcus sp MDB1 at the genus level were all greater than 3.
LEfSe analysis of fungal biomarkers also revealed 25 fungal genera with statistical significance in different treatments (Figure S8). Eight, five, three, seven, and two biomarker fungi were detected in Con, N1T0, N1T1, N2T0, and N2T1 treatments, respectively. The main discriminant group of the N1T1 group, the unidentified LDA value of the Strophariaceae family and its genus in the Basidiomycota phylum, was greater than 4. In the N2T1 group, the LDA value of the Aspergillaceae family and the Penicillium genus under its family was greater than 3.5, which became a significant biomarker species in the treatment group.

3.5. Functional Prediction of Microbial Communities

The FAPROTAX annotation (Figure 2a) showed that the functional groups related to the nitrogen cycle mainly included nitrification (9.87–14.13%), aerobic ammonia oxidation (8.44–12.23%), nitrate reduction (1.61–1.89%), and nitrogen fixation (0.53–3.55%). Compared with T0, the relative abundance of nitrification, aerobic ammonia oxidation, and nitrate reduction functional groups in N1T1 treatment was significantly increased by 43.1%, 44.96%, and 17.74%, respectively. The nitrogen fixation and nitrate reduction functional groups were significantly increased by 8.47% and 6.95% under N2T1 treatment. Under T1 conditions, N2 treatment significantly increased the abundance of nitrogen-fixing functional groups compared with N1 treatment.
The functional groups related to the carbon cycle (average relative abundance higher than 1%) were mainly chemoheterotrophy (26.56–31.83%), oxidative heterotrophic (23.20–27.55%), and aromatic compound degradation (1.72–4.76%). Compared with T0, the degradation of chemoheterotrophy, oxyheterotrophy, and aromatic compounds in N1T1 treatment decreased by 13.12%, 12.35%, and 14.48%, respectively, and increased by 1.31%, 7.78%, and 28.35%, respectively, under N2T1 treatment. Under T1 conditions, N2 treatment reduced the abundance of chemoheterotrophic functional groups and increased the abundance of oxidative heterotrophic and aromatic compound degradation functional groups compared with N1 treatment.
A similar analysis was performed using the FUNGuild database (Figure 2b), and the predictive functional types were divided into pathological, symbiotic, and saprophytic nutritional types by nutritional methods. Among them, fungal parasites–plant pathogens–plant saprophytic bacteria (20.73–53.09%) and plant pathogens (1.35–3.26%) are the dominant functional types of the pathotrophic type (average relative abundance is higher than 1%). The relative abundance of fungal parasites–plant pathogens–plant saprophytic bacteria in the N1T1 group was significantly lower than that in the N1T0 group by 60.96%. Under the condition of T1, the relative abundance of plant pathogens decreased by 18.89% in the N1 treatment and significantly decreased by 53.27% in the N2 treatment.
Animal parasites–faecalis saprophytic bacteria–endophytes–fungal parasites–plant pathogens–undefined saprophytic bacteria–wood saprophytic bacteria (0.86–2.03%) were the dominant functional types of symbiotic nutrition (average relative abundance was higher than 1%). Under the condition of T1, the relative abundance of N1 treatment increased by 135.38%, while the relative abundance of N2 treatment increased by 136.26%.
Fecal saprophytic bacteria–plant saprophytic bacteria–soil saprophytic bacteria (0.61–14.12%), endophytic bacteria–plant saprophytic bacteria–undefined saprophytic bacteria (1.69–3.84%), and plant pathogens–plant saprophytic bacteria–undefined saprophytic bacteria (1.72–3.23%) were the dominant functional types of saprophytic trophic type (average relative abundance was higher than 1%). Among them, the relative abundance of fecal saprophytic bacteria–plant saprophytic bacteria–soil saprophytic bacteria increased by 2195.41% in N1 treatment under T1 conditions and increased by 436.10% in N2 treatment. Endophyte–plant saprophytic bacteria–undefined saprophytic bacteria also had a similar trend. Under T1 conditions, the relative abundance of N1 treatment increased by 124.75%, and the relative abundance of N2 treatment increased by 78.08%. The plant pathogen–plant saprophytic bacteria–undefined saprophytic bacteria showed a completely different phenomenon. Under T1 conditions, the relative abundance of N1 treatment decreased by 37.85%, while the relative abundance of N2 treatment decreased by 21.62%.

3.6. The Effects of Trichoderma Combined with Nitrogen Fertilizer Treatment on Plant Leaf Nutrients, Plant Stem Nutrients, and Grain Nutrients and Their Relationship with Microbial Diversity and Richness Were Analyzed

In order to explore the effect of Trichoderma and nitrogen fertilizer on the nutrient content of each part of the plant, the contents of total nitrogen, total potassium, and total phosphorus in leaves, stems, and grains were analyzed in detail (Table 2). Under N1 conditions, T1 treatment resulted in a decrease in total nitrogen, total potassium, and total phosphorus content in leaves by 3.16%, 7.16%, and 10.00%, respectively. On the contrary, under the N2 condition, T1 treatment significantly increased the contents of total nitrogen, total potassium, and total phosphorus in leaves by 21.37%, 34.04%, and 63.64%, respectively. For stems, under the condition of N1, T1 treatment also led to a downward trend in total nitrogen, total potassium, and total phosphorus content, with a decrease of 15.41%, 39.24%, and 50.00%, respectively, indicating that the effect of Trichoderma on stem nutrients was similar to that of leaves when nitrogen fertilizer was insufficient. However, under the condition of N1, the contents of total nitrogen, total potassium, and total phosphorus in grains showed an upward trend, with an increase of 9.60%, 10.33%, and 5.26%, respectively. Under the condition of N2, this trend was more obvious. T1 treatment increased the contents of total nitrogen, total potassium, and total phosphorus in grains by 31.26%, 37.21%, and 40.00%, respectively, which further confirmed the positive effect of Trichoderma on grain nutrient accumulation when nitrogen fertilizer was sufficient.
Based on the structural equation model (Figure 3), by converting multiple variables into a factor and explaining the influence relationship between variables, the influence efficiency quantity is analyzed in depth through the standardized path coefficient. Among them, bacterial chao1 and bacterial ACE constitute bacterial richness factor, bacterial Simpson and bacterial Shannon constitute bacterial diversity factor, fungal chao1 and fungal ACE constitute fungal richness factor, fungal Simpson and fungal Shannon constitute fungal diversity factor, leaf nitrogen content, leaf potassium content, and leaf phosphorus content constitute plant leaf factor, stem nitrogen content, stem potassium content, and stem phosphorus content constitute plant stem factor, and grain nitrogen content, grain potassium content, and grain phosphorus content constitute grain factor. The relative abundance of Mortierella, Penicillium, and Acremonium constituted beneficial fungal factors, while the relative abundance of Ophiosphaerella and Gaeumannomyces constituted harmful fungal factors. The results showed that plant leaves had a significant positive effect on grain (r = 0.686, p < 0.01), and plant stems had a significant negative effect on grain (r = −0.534, p < 0.01). Bacterial richness had a significant negative effect on plant leaves (r = −0.35), and fungal richness had a significant negative effect on plant leaves (r = −0.575).

4. Discussion

4.1. Effects of Nitrogen Fertilizer Combined with Trichoderma on Microbial Community Composition in Maize Rhizosphere Soil

Proteobacteria, Acidobacteria, and Chloroflexi were the main dominant bacteria in the phylum-level analysis of the bacterial community. Under the T0 condition, the abundance of Proteobacteria increased with the increase in nitrogen fertilizer concentration, which may be related to the direct promotion of nitrogen fertilizer on the growth of some Proteobacteria [17,18]. Under the condition of T1, the abundance of Proteobacteria decreased, which indicated that Trichoderma may directly inhibit the growth of Proteobacteria [19] or the regulation of Trichoderma in the soil environment affected the proliferation of Proteobacteria. Proteobacteria are the main carbon users in the soil. The decrease in their abundance means that the competitive consumption of carbon is reduced, resulting in more carbon being converted into stable organic matter. This transformation not only enhances the carbon sequestration capacity of the soil but also reduces the carbon emissions of the farmland system. The abundance of Acidobacteria and Chloroflexi was low under T0 treatment, but increased under T1 treatment, which was consistent with the growth of ACE index under the same conditions, indicating that Trichoderma may promote the decomposition of organic matter in soil and improve the effectiveness of nutrients [20]. Especially under saline–alkali conditions, Trichoderma can regulate the nutrient absorption and bacterial community structure and function in rhizosphere soil and promote the absorption of nutrients from saline soil [21]. It provides more suitable living conditions for Acidobacteria and Chloroflexi. It was also found that the relative abundance of Proteobacteria, Firmicutes, Cyanobacteria, and Verrucomicrobia increased under high nitrogen conditions but decreased under T1 conditions. At the same time, the decrease in the Shannon index and the increase in the Simpson index also seemed to verify this inhibition phenomenon. This may mean that Trichoderma affects the absorption and fixation of nitrogen fertilizer under saline–alkali conditions in semi-arid areas or regulates the bacterial community related to the nitrogen cycle [22]. PCoA analysis at the gate level based on the Bray–Curtis distance showed that there was a significant difference between T1 treatment and T0 treatment (Figure S5a), which once again echoed the view that the application of Trichoderma may have a positive impact on the bacterial community structure by improving the soil environment and promoting the growth of beneficial microorganisms [23].
Saline–alkali stress can hinder the absorption of water by plant roots, resulting in the destruction of osmotic pressure, cell water balance, and nutrient absorption, and affect the viability of microorganisms by changing the osmotic balance. At the genus level, the application of Trichoderma can create a more suitable microhabitat for functional bacteria closely related to the growth and development of maize by adjusting soil microecology, soil pH, organic matter content, and other soil physical and chemical properties. Further, the microbial community enhances soil structure and water-holding capacity and helps to alleviate salt stress by promoting ion balance in plant cells [24]. Such environmental changes may help to stimulate the growth of bacterial genera such as Lysobacter and Sphingomonas. These genera have been considered to be microorganisms that enhance plant growth and health functions. Among them, Lysobacter belongs to γ-Proteobacteria and secretes biomass-degrading enzymes and plant growth-promoting substances [25], which contribute to the growth of plants such as corn. Sphingomonas has a positive effect on plant growth and can produce plant growth hormone, which may indirectly affect maize growth [26]. Trichoderma may have a positive effect on the bacterial community involved in nitrogen fixation or transformation. For example, Nitrospira, as a nitrifying bacterium, participates in the nitrification process and oxidizes ammonia to nitrite and nitrate, which is of great significance for improving the utilization efficiency of nitrogen fertilizer [27]. PCoA analysis at the genus level showed that the N1T1 group and the N1T0 group had higher clustering similarity (Figure S5b), which indicated that Trichoderma may play a key role in maintaining the stability of bacterial community under low-nitrogen conditions. In the PCoA analysis at the genus level, the variation explained by the first and second principal axes was lower than that at the phylum level, which may mean that the response of bacterial communities at different genus levels to nitrogen fertilizer and Trichoderma was more diverse and complex.
In the analysis of the phylum level of the fungal community, Basidiomycota showed a positive response to the addition of Trichoderma, and the addition of Trichoderma significantly promoted the abundance of Basidiomycota. This not only indicates the key role of basidiomycetes in organic matter decomposition and nutrient cycling [28] but also reflects that Trichoderma may indirectly improve the competitive advantage of Basidiomycota by affecting soil microbial community structure. This is in line with the phenomenon of ACE index growth. For the whole fungal community (Figure S3), the application of Trichoderma significantly regulated its structure, which may indicate that the application of Trichoderma can change the response of the fungal community to nitrogen fertilizer. For example, under N1T1 conditions, the higher abundance of Chytridiomycota may be related to the regulation of Trichoderma on soil conditions.
Further analysis of the fungal community at the genus level, similar to the bacterial community, showed a unique response pattern to T1 treatment. In the environment with low-nitrogen fertilizer application levels, the application of Trichoderma helped maintain the stability of the fungal community, which can also be seen from the significant clustering relationship between the N1T1 and N1T0 treatment groups. From the results of PCoA analysis, T1 treatment had a significant effect on the rhizosphere fungal community structure. Especially in the gate-level analysis (Figure S6a), the contribution rate of the first principal axis was as high as 51.5%, and it was still as high as 49.1% in the genus-level analysis (Figure S6b), which confirmed that the variation of rhizosphere microbial community structure was mainly driven by the interaction between Trichoderma and nitrogen fertilizer.
Under different nitrogen fertilizer levels, the appropriate combination of Trichoderma and nitrogen fertilizer has a significant promoting effect on beneficial fungi and an inhibitory effect on harmful fungi. Under the condition of N1, T1 treatment significantly increased the abundance of three beneficial genera of Mortierella, Penicillium, and Acremonium. Mortierella interacted with plants as a synergist to promote plant growth but decreased significantly under N2 conditions. The promotion effect of Trichoderma on Penicillium under N2 conditions is still significant, increasing by 125.54%. Penicillium produces metabolites beneficial to plant growth, such as penicillin, and inhibits the growth of other pathogens [29], thus indirectly protecting crops [30]. Acremonium can form a symbiotic relationship with plants, enhance plant resistance to pests and diseases, and promote plant growth [31]. This finding suggests that Trichoderma may promote the growth and activity of these beneficial fungi by secreting auxin, enzymes, and other bioactive substances [32], thereby improving soil nutrient cycling and plant nutrient uptake in a low-nitrogen environment and improving salt stress and its interaction with beneficial soil microorganisms [33]. Under N2 conditions, the positive effect of T1 treatment on beneficial fungi still existed, but the growth rate decreased.
For harmful fungi, two crop harmful fungi, Ophiosphaerella and Gaeumannomyces, were screened. Among them, Ophiosphaerella can penetrate plant tissues by making special structures to form diseases [34]; in maize, the genus Gaeumannomyces may cause root rot, resulting in poor plant growth or decreased yield [35]. Through these mechanisms, Trichoderma can inhibit the growth of harmful fungi and reduce their negative effects on crops.

4.2. Effects of Nitrogen Fertilizer Combined with Trichoderma on Microbial Differences and Markers in Maize Rhizosphere Soil

By observing the number of OTUs in different treatment groups (Figure S4), it was found that the N1T1 group showed the highest number of OTUs, indicating that the interaction between Trichoderma and nitrogen fertilizer may significantly increase the diversity of bacteria, and its unique marker Pyrinomonadaceae may be related to plant hormone production [36]. Compared with the T0 treatment, the increase in bacterial community richness in the T1 treatment group reflects that the metabolites of Trichoderma may be beneficial to the improvement of the soil environment [37], which is consistent with previous studies and provides more suitable growth conditions for the bacterial community. The N1 treatment group showed a higher number of common OTUs, which was significantly higher than the N2 treatment group. The microbial diversity under the N1 condition is high and stable, which may be related to the enhancement of microbial activities such as biological nitrogen fixation. Alphaproteobacteria, a unique marker of the N1T0 group, played a role in fixing nitrogen or converting inorganic nitrogen into available forms of plants. There were significant differences in the number of biomarker bacteria in the N2T1 group, suggesting that different nitrogen levels may cause different selection pressures on the rhizosphere microflora [38,39]. The high LDA values of Thermoleophilia and Solirubrobacteraceae, the unique markers of the N2T1 group, indicate that they exhibit growth advantages under N2 conditions, which may be related to their metabolic pathways and nutrient acquisition strategies. At the same time, its unique marker, Blastococcus, is involved in the process of nitrogen fixation or transformation [40].
In the process of detailed analysis of fungal community structure, we found that although the number of OTUs observed overall was not very rich, in our experimental design, the changing trend between different treatment groups has shown significant differences. In particular, under the N1T1 combination treatment, the number of OTUs reached the highest point, indicating that the combined use of appropriate nitrogen fertilizer and Trichoderma had a positive impact on the diversity of fungal communities, thereby promoting its richness. Its unique marker, Strophariaceae, has specific ecological adaptability under low-nitrogen conditions, such as the formation of mycorrhiza or the production of plant growth-stimulating substances [41,42]. In contrast, the N2T0 group showed a significant lag in the number of OTUs, which may be related to the negative effects of saline–alkali and high nitrogen levels on soil pH and organic matter content [43]. This effect may limit the further development of the fungal community, but it is worth noting that the addition of Trichoderma seems to be able to alleviate this negative impact by improving soil structure and providing the necessary organic nutrition, which has a certain protective effect on the healthy development of the fungal community. The unique marker of N2T1, Penicillium of the Aspergillaceae family, is a significant biomarker species, which echoes the beneficial species promoted by the Trichoderma screened above.

4.3. Prediction of Nitrogen Fertilizer Combined with Trichoderma on Microbial Micro-Function in Maize Rhizosphere Soil

Based on the analysis of the prediction of rhizosphere soil microbial function, we found that the sensitivity and specificity of Trichoderma treatment to soil microbial response were different. In order to explore the effect of Trichoderma on the functional group of the soil bacterial community, the FAPROTAX database was used to focus on the nitrogen cycle and carbon cycle. Three dominant functional groups were related to the nitrogen cycle (nitrification, aerobic ammonia oxidation, and nitrate reduction), and their relative abundance increased significantly under N1T1 treatment. This finding suggests that T1 treatment may promote these important processes related to nitrogen transformation under saline–alkali conditions. Nitrification and aerobic ammonia oxidation are two key steps in the nitrogen cycle, which convert ammonia to nitrite and nitrate and then provide the necessary nitrogen source for plant growth [44]. Under N1 conditions, T1 treatment resulted in a 43.1% and 44.96% increase in the relative abundance of nitrification and aerobic ammonia oxidation functions, respectively, which may be attributed to the positive effects of secretions or altered microbial environments produced by Trichoderma on nitrifying bacteria and aerobic ammonia-oxidizing bacteria. However, higher potential nitrification increases the content of nitrate nitrogen that can be absorbed and utilized by plants, thereby promoting plant growth but increasing the risk of nitrogen loss [45,46]. In addition, the relative abundance of nitrate reduction function was also significantly increased in the T1 treatment group, which may indicate a promoting effect on the important denitrification process of nitrate reduction to nitrite. Efficient nitrate reduction function means that plants can more effectively utilize nitrogen in the soil and alleviate the inhibition of salinity on nitrogen absorption, thereby reducing nitrogen loss [47]. Enhancing biological nitrogen fixation can significantly improve agricultural systems and optimize the environmental and economic costs associated with nitrogen supply [48].
Contrary to the nitrogen cycle, T1 treatment seemed to inhibit the dominant functional groups related to the carbon cycle under N1 conditions, including chemical heterotrophic, good oxidation heterotrophic, and aromatic compound degradation, and the relative abundance of these functions decreased by 13.12%, 12.35%, and 14.48%. These results may reflect the inhibitory effect of Trichoderma on organic carbon-degrading microorganisms in soil, thus achieving carbon sequestration. However, the exact mechanism of carbon sequestration may be more complicated. Trichoderma may enhance the stability of soil aggregates through secretions, thereby reducing the decomposition rate of organic carbon and facilitating long-term storage of carbon, improving the structure of saline–alkali soil and increasing soil organic matter content, indirectly promoting carbon sequestration [49]. It may also be because Trichoderma itself, as a decomposer, competes with these functional groups for resources or affects the metabolic activities of microorganisms by changing soil chemical properties. In addition, the relative abundance of the aromatic compound degradation functional group is reduced so that the degradation rate of aromatic compounds, which are an important part of soil organic carbon, may contribute to the long-term fixation of carbon [50]. The increase in soil carbon sequestration leads to a decrease in carbon dioxide emissions, slows global warming, and helps increase soil organic carbon levels [51,52].
The FUNGuild database was used to screen and classify the functional types of soil fungi. The results showed that T1 treatment significantly reduced the function of pathotrophic microorganisms, especially the relative abundance of fungal parasites–plant pathogens–plant saprophytic bacteria, which decreased by 60.96% under N1 conditions. This result indicates that Trichoderma may inhibit pathogen growth by competing or producing inhibitory substances, thereby reducing the pathogen burden of plant roots and helping to improve plant health and growth. Animal parasites–faecalis saprophytic bacteria–endophytes–fungal parasites–plant pathogens–undefined saprophytic bacteria–wood saprophytic bacteria, as a symbiotic nutritional microbial functional group, are the bearers of beneficial symbiotic relationships and show a significant increase in abundance under T1 treatment, especially under N1 conditions. It was increased by 135.38%. This suggests that T1 may promote the establishment of beneficial symbiotic relationships with plants, such as mycorrhizal fungi and endophytes, which may enhance plant nutrient absorption capacity and resistance to environmental stresses such as drought and salinity [53]. Saprotrophic microorganisms mainly exist by decomposing organic matter, showing different response modes under T1 treatment. The abundance of fecal saprophytic bacteria–plant saprophytic bacteria–soil saprophytic bacteria increased significantly by 2195.41% under N1 conditions, indicating that T1 greatly promoted the decomposition of organic matter and nutrient cycling. The abundance of plant pathogens–plant saprophytic bacteria–undefined saprophytic bacteria decreased under T1 treatment, which may indicate that T1 played a selective role in regulating soil microbial community structure. By reducing the number of pathogenic microorganisms, increasing beneficial symbiotic microorganisms, and promoting the decomposition of organic matter, the application of Trichoderma helps to build a more stable and more productive agricultural ecosystem and alleviate the abiotic stress caused by drought and salinity.

4.4. The Effects of Trichoderma Combined with Nitrogen Fertilizer Treatment on Plant Leaf Nutrients, Plant Stem Nutrients, and Grain Nutrients and Their Relationship with Microbial Diversity and Richness Were Analyzed

Nitrogen fertilizer is an indispensable nutrient cornerstone for plant growth, and its deficiency often restricts the uptake and transformation of key nutrients such as nitrogen, potassium, and phosphorus. The mechanism of action of Trichoderma may involve competition with plant rhizosphere nutrients or by secreting specific metabolites, thereby affecting the nutrient storage site of plants, resulting in a decrease in nutrient content in leaves [32]. This phenomenon reveals that in an environment with sufficient nitrogen supply, Trichoderma can significantly improve the absorption efficiency of nitrogen, potassium, and phosphorus by plants, thereby enriching the nutrient reserves in leaves. The possible mechanisms of this effect include the improvement of soil structure, the improvement of soil fertility, or the direct promotion of plant growth and nutrient absorption through the establishment of symbiotic relationships [54]. Following further observation of the stems, we found that even under low-nitrogen conditions, Trichoderma treatment also led to a downward trend in total nitrogen, total potassium, and total phosphorus content, echoing the response of the leaves. However, in the grain part, whether under N1 or N2 conditions, Trichoderma treatment promoted the increase in total nitrogen, total potassium and total phosphorus content, especially under the condition of sufficient nitrogen fertilizer N2, this upward trend was more significant. This finding underscores the positive effect of Trichoderma on grain nutrient accumulation under sufficient nitrogen conditions, which may be due to Trichoderma improving the photosynthetic efficiency of plants, thereby promoting grain formation and nutrient accumulation [55].
Based on the structural equation model, considering that leaves are the main place where photosynthesis occurs, their functional status directly determines the efficiency of plant production of organic matter. This result highlights the positive correlation between leaf and grain quality (r = 0.686, p < 0.01). On the contrary, there was a significant negative correlation between stem and grain (r = −0.534, p < 0.01). This phenomenon may be due to the fact that when the stem development is too strong, it may not only compete with the leaves for light [56] but also participate in the competition for limited photosynthetic products in the plant, thus indirectly affecting the healthy growth of the grain.
For the richness and diversity of microbial communities, our study shows that they have a certain negative impact on leaves. The proliferation of microorganisms may compete for necessary nutrients during the critical period of leaf growth, affecting the absorption and utilization of nutrients by leaves. We found that the saline–alkali soil environment may change the negative effects of harmful fungi on plants, or the effects of harmful fungi are not directly on stems and leaves [57]. The effect of microbial diversity on plant leaves is complex and changeable, suggesting that we need to consider the potential impact of strains on leaf nutritional status in future research.

5. Conclusions

The results of this study clearly showed that the application of Trichoderma increased the abundance of beneficial bacteria, such as Penicillium, and inhibited the abundance of harmful bacteria, such as Ophiosphaerella. From the perspective of microbial function prediction, Trichoderma may promote the soil nitrogen cycle through the nitrification process and nitrogen fixation process under different nitrogen application levels, as well as promote soil carbon sequestration by weakening soil carbon degradation. In particular, N1T1 treatment not only increased the species richness of bacterial communities but also effectively improved soil fertility and promoted crop growth and nutrient uptake under saline–alkali conditions in semi-arid areas by optimizing soil microbial structure and rhizosphere microecology.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14102340/s1, Figure S1: The alpha diversity index statistics of bacteria and fungi in rhizosphere soil under the condition of Trichoderma combined with different nitrogen application rates; Figure S2: The heat map was clustered according to the similarity of bacterial community composition between treatments and horizontally arranged according to the clustering results; Figure S3: The heat map was clustered according to the similarity of fungal community composition between treatments and horizontally arranged according to the clustering results; Figure S4: Venn diagram of OTU level in maize rhizosphere soil under different treatments; Figure S5: Based on the Bray–Curtis distance, the principal coordinate analysis; Figure S6: Based on the Bray–Curtis distance, the principal coordinate analysis; Figure S7: The bacterial biomarkers of each treatment were determined by linear discriminant analysis (LDA) with a threshold of 3.0; Figure S8: The fungal biomarkers for each treatment were determined by linear discriminant analysis (LDA) with a threshold of 3.0.

Author Contributions

Conceptualization, Y.L. and J.C.; methodology, Y.L. and K.Y.; software, Y.L.; validation, Y.L., J.C. and J.K.; formal analysis, Y.L., J.C., J.K., W.Z., K.Y. and J.F.; investigation, Y.L., J.C., J.K., W.Z., K.Y. and J.F.; resources, W.Z.; data curation, Y.L., J.C., J.K., W.Z., K.Y. and J.F.; writing—original draft preparation, J.C.; writing—review and editing, Y.L.; visualization, Y.L.; supervision, J.F. and J.K.; project administration, Y.L.; funding acquisition, J.F. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Strategic Priority Research Program of the Chinese Academy of Science (Grant No. XDA28130103); the Heilongjiang Province Agricultural Scientific Attack Project; and the Introduced Talent Person of Heilongjiang Bayi Agricultural University (Grant No. XYB201901).

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Acknowledgments

Part of this work was carried out by using the resources of the Agricultural College of Heilongjiang Bayi Agricultural University and Heilongjiang Key Laboratory of Modern Agricultural Cultivation Technology and Crop Germplasm Improvement.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Cluster tree structure and relative abundance of microorganisms in maize rhizosphere soil under different treatments ((a): phylum level of bacteria, (b): genus level of bacteria, (c): phylum level of fungi, (d): genus level of fungi). The relative abundance of beneficial and harmful fungi at the genus level ((c): beneficial fungi, (d): harmful fungi) was compared by functional screening, (e): The relative abundance of beneficial fungi at the genus level was compared by functional screening, (f): The relative abundance of harmful fungi at the genus level was compared by functional screening. Con (control), N1T0 (nitrogen 60), N1T1 (nitrogen 60 + Trichoderma), N2T0 (nitrogen 300), N2T1 (nitrogen 300 + Trichoderma).
Figure 1. Cluster tree structure and relative abundance of microorganisms in maize rhizosphere soil under different treatments ((a): phylum level of bacteria, (b): genus level of bacteria, (c): phylum level of fungi, (d): genus level of fungi). The relative abundance of beneficial and harmful fungi at the genus level ((c): beneficial fungi, (d): harmful fungi) was compared by functional screening, (e): The relative abundance of beneficial fungi at the genus level was compared by functional screening, (f): The relative abundance of harmful fungi at the genus level was compared by functional screening. Con (control), N1T0 (nitrogen 60), N1T1 (nitrogen 60 + Trichoderma), N2T0 (nitrogen 300), N2T1 (nitrogen 300 + Trichoderma).
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Figure 2. Rhizosphere soil bacterial community function (soil carbon cycle and nitrogen cycle-related dominant tube energy group) and fungal community function prediction (pathotrophic, symbiotic, and saprophytic) under different treatments ((a): bacteria, (b): fungi). Con (control), N1T0 (nitrogen 60), N1T1 (nitrogen 60 + Trichoderma), N2T0 (nitrogen 300), N2T1 (nitrogen 300 + Trichoderma).
Figure 2. Rhizosphere soil bacterial community function (soil carbon cycle and nitrogen cycle-related dominant tube energy group) and fungal community function prediction (pathotrophic, symbiotic, and saprophytic) under different treatments ((a): bacteria, (b): fungi). Con (control), N1T0 (nitrogen 60), N1T1 (nitrogen 60 + Trichoderma), N2T0 (nitrogen 300), N2T1 (nitrogen 300 + Trichoderma).
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Figure 3. Structural equation model (SEM) shows the direct effects of bacteria, fungal richness and diversity, and beneficial and harmful fungi on plant leaves, plant stems, and grains. Green and brown represent positive and negative effects. CMIN/DF: chi-square degree of freedom ratio, which is used to evaluate the goodness of fit between the model and the data. GFI: goodness-of-fit index, mainly using the determination coefficient and regression standard deviation to test the fitting degree of the model to the sample observations. CFI: comparing the fitting index, the size of the sample capacity is basically unaffected by it, which can better reflect the situation of the model and is an ideal relative fitting index. ** means p < 0.01 level difference, *** means p < 0.001 level difference.
Figure 3. Structural equation model (SEM) shows the direct effects of bacteria, fungal richness and diversity, and beneficial and harmful fungi on plant leaves, plant stems, and grains. Green and brown represent positive and negative effects. CMIN/DF: chi-square degree of freedom ratio, which is used to evaluate the goodness of fit between the model and the data. GFI: goodness-of-fit index, mainly using the determination coefficient and regression standard deviation to test the fitting degree of the model to the sample observations. CFI: comparing the fitting index, the size of the sample capacity is basically unaffected by it, which can better reflect the situation of the model and is an ideal relative fitting index. ** means p < 0.01 level difference, *** means p < 0.001 level difference.
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Table 1. The design of experimental processing.
Table 1. The design of experimental processing.
TrichodermaNo Trichoderma Agent (T0)Trichoderma Agent (T1)
Nitrogen Level
60 kg N ha−1 (N1)N1T0N1T1
300 kg N ha−1 (N2)N2T0N2T1
Table 2. The nitrogen, potassium, and phosphorus content in different parts of corn plants treated differently. Different lowercase letters in the figure indicate that there are significant differences between different treatments of the same plant organ (p < 0.05). Con (control), N1T0 (nitrogen 60), N1T1 (nitrogen 60 + Trichoderma), N2T0 (nitrogen 300), N2T1 (nitrogen 300 + Trichoderma).
Table 2. The nitrogen, potassium, and phosphorus content in different parts of corn plants treated differently. Different lowercase letters in the figure indicate that there are significant differences between different treatments of the same plant organ (p < 0.05). Con (control), N1T0 (nitrogen 60), N1T1 (nitrogen 60 + Trichoderma), N2T0 (nitrogen 300), N2T1 (nitrogen 300 + Trichoderma).
Plant
Organs		TreatmentsTotal Nitrogen
(g/kg)		Total Potassium
(mg/kg)		Total Phosphorus
(g/kg)
LeafCon17.20 e4555.57 d0.13 b
N1T016.16 d5793.80 b0.10 c
N1T115.65 c5378.82 c0.09 d
N2T015.30 b5727.31 b0.11 c
N2T118.57 a7676.90 a0.18 a
StemCon3.08 c7312.98 c0.03 a
N1T03.05 d8472.85 a0.02 a
N1T12.58 e5148.07 d0.01 a
N2T03.81 b7926.38 b0.02 a
N2T14.21 a3926.60 e0.08 a
GrainCon12.08 d1752.34 e0.16 d
N1T013.64 c1967.23 c0.19 c
N1T114.95 a2170.43 b0.20 b
N2T010.75 e1929.60 d0.15 e
N2T114.11 b2647.65 a0.21 a
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Li, Y.; Cui, J.; Kang, J.; Zhao, W.; Yang, K.; Fu, J. Trichoderma Rhizosphere Soil Improvement: Regulation of Nitrogen Fertilizer in Saline–Alkali Soil in Semi-Arid Region and Its Effect on the Microbial Community Structure of Maize Roots. Agronomy 2024, 14, 2340. https://doi.org/10.3390/agronomy14102340

AMA Style

Li Y, Cui J, Kang J, Zhao W, Yang K, Fu J. Trichoderma Rhizosphere Soil Improvement: Regulation of Nitrogen Fertilizer in Saline–Alkali Soil in Semi-Arid Region and Its Effect on the Microbial Community Structure of Maize Roots. Agronomy. 2024; 14(10):2340. https://doi.org/10.3390/agronomy14102340

Chicago/Turabian Style

Li, Yicong, Jianming Cui, Jiarui Kang, Wei Zhao, Kejun Yang, and Jian Fu. 2024. "Trichoderma Rhizosphere Soil Improvement: Regulation of Nitrogen Fertilizer in Saline–Alkali Soil in Semi-Arid Region and Its Effect on the Microbial Community Structure of Maize Roots" Agronomy 14, no. 10: 2340. https://doi.org/10.3390/agronomy14102340

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