Effects of Organic Fertilizer Replacing Some Nitrogen Fertilizers on the Structure and Diversity of Inter-Root Soil Fungal Communities in Potato

Abstract

The aim of this study was to investigate the effects of organic fertilizer replacing part of the nitrogen fertilizers on the structure and diversity of the inter-root soil fungal communities of potatoes. By carrying out a field trial in Gaoquan Village, Tuanjie Town, Dingxi City, Gansu Province, the main potato-producing area in China, the optimal proportion of organic fertilizer to replace nitrogen fertilizer was determined to provide a scientific basis for the rational use of organic fertilizer to improve the structure and function of soil fungal communities. The experiment was laid out with six treatments: CK (no nitrogen fertilizer, phosphate and potash fertilizer applied), CF (nitrogen fertilizer alone, control), T1 (25% replacement of nitrogen fertilizer), T2 (50% replacement of nitrogen fertilizer), T3 (75% replacement of nitrogen fertilizer), and T4 (100% replacement of nitrogen fertilizer). A systematic study of the inter-root soil fungal community structure, diversity, and soil physicochemical properties during potato harvesting was conducted using high-throughput sequencing technology. The results show that the organic fertilizer replacing part of the nitrogen fertilizer significantly increased the content of alkaline dissolved nitrogen, quick-acting potassium, quick-acting phosphorus, and organic matter in the inter-root soil of the potatoes, and significantly reduced the pH value of the soil. There was a trend of decreasing soil fungal abundance and a significant decrease in the Alpha diversity of the soil fungi. The treatment groups in the soil had as their core fungi Acomycota, Mortierellomycota, Basidiomycota, and others. The organic fertilizers replacing the nitrogen fertilizers significantly altered the structural composition of the inter-root soil fungal community of the potatoes, and increased the differential fungi in the soil. The number of functionally diverse and complex fungi in the soil gradually increased, and the function of the fungal community gradually changed from Singularity to diversification and complexity. A redundancy analysis showed that the soil pH was the main environmental factor affecting the inter-root soil fungal communities of potatoes under organic fertilizer replacing N fertilizer.

1. Introduction

China is one of the world’s major potato-producing areas, and the potato industry plays an important role in promoting the development of China’s agricultural and rural economy. Fertilization can promote the growth and reproduction of microorganisms in the soil by adding the nutrients and organic matter needed by crops to the soil, thus affecting the structure and function of the soil microbial community [1]. The current application of organic fertilizers is an effective way to improve soil fertility and increase nutrient vitality, purify the soil environment, and ensure high yields and the quality of crops based on a variety of amino acids and humic substances [2,3]. Fertilization treatments affect the soil microbiota, significantly influencing the relative abundance of certain bacterial and fungal taxa in the soil, which in turn affects soil health, nutrient cycling, and crop productivity [4]. Organic fertilizers contain a wide range of organic nutrients, including organic matter, humus, and beneficial microorganisms, which can be provided in a comprehensive and balanced manner. Nutrients are needed at different growth stages. The rich nutrients and organic matter in organic fertilizers can effectively improve soil fertility and fertilization [5,6,7]. The application of organic fertilizer can effectively improve the structure and composition of the soil microbial community, which in turn changes the soil microbiota and increases the microbial community stability of the soil [8,9]. Soil fungi have important ecological functions, such as decomposition and parasitism, and their involvement in soil humus formation, nutrient cycling, the stabilization of organic matter, the formation of soil aggregates, and the suppression of plant pathogens are key indicators for assessing soil fertility and health [10,11]. The results of a study conducted by Sileshi et al. [12] in sub-Saharan Africa, including Ethiopia, showed that a mixture of organic and inorganic fertilizers resulted in higher crop yields compared to manure or inorganic fertilizers alone. Li [13] and others found that the application of organic fertilizers has a significant effect on the composition and activity of soil microbial communities, which can change the types of metabolites and the abundance of microorganisms in the soil, thus helping to maintain the stability of microbial diversity. Ma [14] and others found that the use of organic fertilizers to replace some chemical fertilizers can enhance the nutrient levels in the soil and positively affect the diversity of soil microorganisms and their functioning, which in turn contributes to an increase in crop yields. Khan [15] and others found that organic and inorganic fertilizers can promote the effective regulation of nitrogen fertilizers by soil microorganisms to ensure the balanced supply of nutrients required by crops, improve fertilizer efficiency from the perspective of fertilizer supply intensity and supply capacity, and thus improve the nitrogen use efficiency of crops. Jin et al. [16] found that the use of organic fertilizers to replace 20% of nitrogen fertilizers significantly increased the diversity index of bacteria in the soil and reduced the number of some fungi. Song Weifeng [17] showed that the proportion of the organic substitution of nitrogen had a significant effect on soil bacterial diversity and community composition, and was able to significantly increase the abundance of some specific bacterial species. Kumar et al. [18] found that organic fertilizers are rich in nutrients, including organic matter, humus, and beneficial microorganisms, and the application of organic fertilizers increases the abundance of soil microorganisms and facilitates the functional diversity of soil microorganisms. Parente [19] found that organic fertilizers increase the organic matter of soil, which in turn improves soil fertility and maintains microbial diversity. Tang [20] and others found that the use of organic fertilizers to replace some of the nitrogen fertilizers can enhance soil function and adjust the structure of the fungal community, changing the structure of the fungal community to enhance the interactions within and between microbial communities.

Overall, organic N substitution affects microbial diversity and community structure, and the trends for the various microbial components are not identical at different levels of organic N substitution. Therefore, it is important to understand the impact of the ratio of organic fertilizer to nitrogen fertilizer on the changing characteristics of the potato soil fungal community. In this paper, based on the positioning test of nitrogen organic substitution at the Gaoquan experimental base in Dingxi, the main potato-production area in China, the experimental design, from a 0 to 100% organic fertilizer substitution rate, is comprehensively analyzed along with the characteristics of the changes in the inter-root fungal community of the potatoes with the organic fertilizer substitution of nitrogen fertilizer. A high-throughput sequencing method is used to explore the changing characteristics of the potato soil fungal communities and the factors that influence them under different proportions of organic fertilizers replacing chemical fertilizers, and to analyze the relationship between soil fungal communities and environmental factors, such as soil pH, organic matter content, and nutrient levels. Thus, this paper provides guidance for soil management and agricultural practice, and further provides a theoretical basis and practical reference for the popularization and application of potato cultivation under organic fertilizer replacing nitrogen fertilizer.

2. Materials and Methods

2.1. Overview of the Study Area

This experimental study was carried out at the Gaoquan Experimental Station of the Gansu Provincial Academy of Agricultural Sciences located in Gaoquan Village, Tuanjie Township, Anding District, Dingxi City (geographic coordinates 35°24′21″ N, 104°34′94″ E). The altitude of the area is 2000 m, the average annual temperature is about 6.8 °C, the average annual precipitation is about 415 mm, and the frost-free period is between 146 and 149 days, which is typical of the northwest loess hilly, semi-arid region. The soil in the test area is mainly of the loess type. The annual precipitation and soil nutrient content of the test site are shown in

Table 1. Precipitation at test site.

Months	January	February	March	April	May	June	July	August	September	October	November	December	Total
Precipitation (mm)	2.2	27	9.3	43.3	53.7	27	67.7	24.8	52.1	61.6	0.6	0.3	369.6

and

Table 2. Basic physical and chemical properties of soil in test area.

Nutrients	Total Nitrogen (g·kg−1)	Total Phosphorus (g·kg−1)	Total Potassium (g·kg−1)	Quick-Acting Nitrogen (mg·kg−1)	Quick-Acting Phosphorus (mg·kg−1)	Quick-Acting Potassium (mg·kg−1)	Organic Matter (g·kg−1)	pH
quantity contained	0.73	0.67	19.90	50.83	15.12	174.81	12.93	8.21

.

2.2. Experimental Design

The experiment was conducted in a randomized block design on the same plot with uniform soil texture and fertility, and six treatments were set up as follows: CK (no N fertilizer, phosphorus and potassium fertilizer), CF (single N fertilizer, control), T1 (25% organic fertilizer replacing N fertilizer), T2 (50% organic fertilizer replacing N fertilizer), T3 (75% organic fertilizer replacing N fertilizer), and T4 (100% organic fertilizer replacing N fertilizer). The organic fertilizer utilized in the experiment was a commercially available product with a minimum organic matter content of 45%, nitrogen content of 1.15%, phosphorus content of 0.7%, and potassium content of 0.5%. The nitrogen fertilizer was urea with a nitrogen content of 46%, the phosphorus fertilizer was calcium superphosphate with a phosphorus content of 12%, and the potassium fertilizer was potassium sulfate with a potassium content of 50%. The nutrient application rates of nitrogen, phosphorus, and potash were 180 kg/hm², 90 kg/hm², and 90 kg/hm², respectively. The K₂O application rate was 60 kg/hm². Each treatment was replicated three times, with a plot area of 45 m². The potato research material variety was “Longshu No. 7”. Before sowing the potatoes, all the fertilizers were applied to the soil at once. The planting method of full-film-covered ridge sowing was adopted, i.e., the ground was fully covered with a film to form a single-row ridge with a width of 60 cm, a height of 15 cm, and a furrow with a width of 40 cm. The potato planting position was located on both sides of the ridge. Sowing usually took place in early May, and harvesting was scheduled for mid-October. Other field management measures followed the usual practices of the local farmers. The nutrient dosage and organic fertilizer N replacement rate for each treatment are tabulated in

Table 3. Nutrient use and organic fertilizer nitrogen replacement rate by treatment.

Treatment	Organic Fertilizer (kg/hm2)	Nitrogen Fertilizer Type (kg/hm2)			Organic Fertilizer on Nitrogen Substitution Rate (%)
		N	P2O5	K2O
CK	0	0	90	60	0
CF	0	180	90	60	0
T1	4000	135	90	60	25
T2	8000	90	90	60	50
T3	12,000	45	90	60	75
T4	16,000	0	90	60	100

.

2.3. Sample Collection

Five potato plants were randomly selected as samples from each plot one week before potato harvest. The soil-shaking method was used to treat these plants: First, the plants were gently shaken to remove large pieces of non-inter-root soil; the portion that did not contain roots. Next, the plants were shaken vigorously to dislodge the soil attached to the roots and drop it onto a sieve (for the portion that was difficult to dislodge, the edges of the sieve could be lightly tapped to assist in the dislodging of the soil). Impurities, such as debris and roots, were then removed from the soil samples using tweezers, then mixed well, and sieved through a 2 mm pore-size sieve. The processed soil samples were placed in sterilized bags and placed in an insulated box along with ice packs for transport back to the laboratory. Upon arrival at the laboratory, these samples were stored in a refrigerator at −80 °C for analysis and determination of soil microorganisms.

2.4. Measurement of Soil Physical and Chemical Indicators

Acidity measurements were conducted using a pH meter (PB-10, Sartorius, Göttingen, Germany) at a water-to-soil ratio of 5:1. The sulfuric acid-potassium dichromate external heating method was employed to determine the organic matter (SOM), while the Kjeldahl method was utilized to ascertain the total nitrogen (TN). The HClO4-H2SO4 method was used to determine the total phosphorus (TP), and the acid digestion-inductively coupled plasma emission spectrometry (ACPES) method was applied to determine the total potassium (TK). The alkaline diffusion method was used to determine the alkaline nitrogen (AN). The sodium bicarbonate leaching method, which employs the use of aluminum antimony colorimetry, was utilized for the determination of the quick-acting phosphorus (AP). Similarly, the ammonium acetate leaching method, which employs flame photometry, was employed for the determination of the quick-acting potassium (AP). Both of these determinations were referenced in the “Determination of Potassium in Forest Soil” and “Soil Agrochemical Analysis” [21].

2.5. High-Throughput Sequencing of Soil Fungi

The total genomic DNA of the microbial community was extracted using a FastPure Soil DNA Isolation Kit (Magnetic bead) (MJYH, Shanghai, China), and then the quality of the extracted genomic DNA was detected using 1% agarose gel electrophoresis, and the DNA concentration was determined using a NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA.) to determine the DNA concentration. The concentration of extracted DNA ranged from 7.92 to 15.43 ng-μL⁻¹. The upstream primer ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and downstream product ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) [22], both of which carry a barcode sequence, were used for PCR amplification of the ITS1 and ITS2 regions, variable regions of the ITS gene. For the variable region, the PCR reaction system was as follows: 10 × Buffer 2 μL, 2.5 mM dNTPs 2 μL, upstream primer (5 uM) 0.8 μL, downstream primer (5 uM) 0.8 μL, rTaq Polymerase 0.2 μL, BSA 0.2 μL, Template DNA 10 ng, and makeup ddH2O to 20 μL. The amplification program was as follows: (1) 1 × (3 min at 95 °C); (2) cycle number × (30 s at 95 °C; 30 s at 55 °C; 45 s at 72 °C); and (3) 10 min at 72 °C, 10 °C until halted by the user (PCR instrument: ABI GeneAmp^® Model 9700 (ABI, CA, USA)). The PCR products were recovered on a 2% agarose gel, purified using a DNA gel recovery and purification kit (PCR Clean-Up Kit: MJYH, Shanghai, China), and quantified using Qubit 4.0 (Thermo Fisher Scientific, USA).

The purified PCR products were used for library construction using a NEXTFLEX Rapid DNA-Seq Kit (Illumina, San Diego, CA, USA): (1) junction linkage; (2) removal of junction self-linking fragments using magnetic bead screening; (3) enrichment of library templates using PCR amplification; and (4) recovery of PCR products by magnetic beads to obtain the final library. Sequencing was performed using the Illumina PE300 platform (Shanghai Meiji Biomedical Technology Co., Ltd., Shanghai, China). The raw data were uploaded to the NCBI SRA database.

2.6. Data Processing

The double-ended raw sequenced sequences were quality controlled using fast [23] (https://github.com/OpenGene/fastp, version 0.19.6, accessed on (8 October 2024)) software, and FLASH [24] (http://www.cbcb.umd.edu/software/flash, the version 1.2.11, accessed on (9 October 2024)) software for splicing, as follows: (1) Filter the bases with a quality value below 20 at the end of the reads, and set a window of 50 bp. If the average quality value within the window is below 20, truncate the back-end bases from the beginning of the window, filter the reads with less than 50 bp after the quality control, and remove the reads containing N bases. (2) Splice (merge) the pairs of reads into a single sequence based on the overlap relationship between the PE reads, with a minimum overlap length of 10 bp. (3) Screen for non-compliant sequences, with a maximum mismatch ratio of 0.2 allowed in the overlap region of the spliced sequences. (4) Distinguish the samples based on the barcode and primers on the first and last ends of the sequences and adjust the sequence orientation; the allowed number of mismatches for the barcode was 0 and the maximum number of primer mismatches was 2. Using Uparse version 7.0.1090 software [25] (http://drive5.com/uparse, accessed on (10 October 2024)), based on a 97% similarity, the sequences after QC splicing were subjected to operational taxonomic unit (OTU) clustering and the elimination of chimeras. The annotated chloroplast and mitochondrial sequences were removed from all the samples. To minimize the impact of the sequencing depth on the subsequent analysis of the Alpha diversity and Beta diversity data, the number of sequences in all the samples was drawn flat to 20,000, after which the average sequence coverage (Good’s coverage) per sample could still be up to 99%. The OTU species taxonomy was annotated using the RDP classifier [26] (https://sourceforge.net/projects/rdp-classifier/, version 2.11) compared to the species classification database (unite8.0/its_fungi), with a confidence threshold of 70%. And the community composition of each sample was counted at different species classification levels. The fungal function prediction analysis was performed using Funguild (version 1.0) software.

Microsoft Excel 2016 software was used to process the raw data and plots, and a one-way ANOVA, as well as significance tests, were performed using SPSS 27.0 statistical analysis software. The sequencing data were analyzed on the Meiji BioCloud platform (www.majorbio.com). The plotted data were processed with Adobeillustrator 2020 software as follows: the mother [27] software (http://www.mothur.org/wiki/Calculators, accessed on (10 October 2024)) was used to calculate the Alpha diversity Chao 1, Shannon index, etc., and a one-way ANOVA was used to analyze the between-group differences in Alpha diversity; the similarity of the microbial community structure among the samples was examined using a thisprincipal coordinate analysis (PCoA) based on Bray-curtis dissimilarity (principal coordinate analysis) to test the similarity of the microbial community structure among the samples, and an LEfSe analysis (linear discriminant analysis effect size) [28] (http://huttenhower.sph.harvard.edu/LEfSe, accessed on (11 October 2024)) (LDA > 3.5, p < 0.05) was performed to identify the fungal taxa with significant differences in abundance between the groups. A distance-based redundancy analysis (db-RDA) was used to investigate the effect of the soil physicochemical indicators on the soil fungal community structure.

3. Results and Analysis

3.1. Analysis of Chemical Properties of Potatoes’ Inter-Root Soil Under Organic Fertilizer Replacing Nitrogen Fertilizer

As shown in

Table 4. Table of soil chemical properties for each treatment.

Treatment	Total Nitrogen (g·kg−1)	Total Potassium (g·kg−1)	Total Phosphorus (g·kg−1)	Alkaline Nitrogen Decomposition (mg·kg−1)
CK	0.770 ± 0.038 b	19.81 ± 0.18 b	0.665 ± 0.003 b	55.17 ± 4.06 c
CF	1.193 ± 0.020 a	20.13 ± 0.21 ab	0.892 ± 0.015 a	58.03 ± 2.65 c
T1	1.213 ± 0.041 a	20.32 ± 0.02 a	0.895 ± 0.027 a	69.65 ± 5.28 b
T2	1.247 ± 0.029 a	20.41 ± 0.08 a	0.882 ± 0.012 a	77.65 ± 2.84 ab
T3	1.260 ± 0.025 a	20.47 ± 0.14 a	0.893 ± 0.003 a	84.70 ± 4.78 a
T4	1.301 ± 0.056 a	20.54 ± 0.22 a	0.897 ± 0.015 a	82.03 ± 1.55 a
Treatment	Quick-acting potassium (mg·kg−1)	Quick-acting phosphorus (mg·kg−1)	Organic matter (g·kg−1)	pH
CK	130.95 ± 5.06 c	16.94 ± 0.68 b	14.59 ± 0.92 c	8.10 ± 0.08 a
CF	140.29 ± 4.39 c	17.02 ± 2.25 b	15.79 ± 1.40 bc	7.89 ± 0.04 b
T1	143.65 ± 8.29 c	23.11 ± 0.38 a	17.53 ± 0.22 ab	7.70 ± 0.04 c
T2	165.72 ± 20.00 bc	25.59 ± 1.55 a	17.31 ± 0.20 ab	7.65 ± 0.06 c
T3	199.16 ± 16.97 ab	27.93 ± 2.51 a	17.71 ± 0.19 ab	7.60 ± 0.03 c
T4	216.29 ± 11.14 a	26.86 ± 1.70 a	18.15 ± 0.27 a	7.56 ± 0.02 c

Data are mean ± standard deviation, n = 3. Different letters in same column indicate significant differences between different fertilization treatments (p < 0.05).

, compared with the CF, the alkaline nitrogen content of each alternative treatment group is significantly increased (p < 0.05); the quick-acting phosphorus content is significantly increased (p < 0.05); and the quick-acting potassium content is significantly increased (p < 0.05) in the T3 and T4 treatment groups. The pH value of each treatment group is significantly reduced (p < 0.05). The organic matter content of the T4 treatment group is significantly increased (p < 0.05). The total nitrogen, phosphorus, and potassium in the soil of each treatment group tends to stabilize without any significant difference. The contents of total phosphorus, total potassium, and total nitrogen are significantly increased (p < 0.05) compared to the CK treatment. The incorporation of organic fertilizers significantly increases the contents of alkaline dissolved nitrogen, quick-acting potassium, quick-acting phosphorus, and organic matter in the inter-root soil of the potatoes, and significantly decreases the pH of the soil.

3.2. OTU Analysis of Inter-Root Soil Fungi of Potatoes Under Organic Fertilizer Replacing Nitrogen Fertilizer

A sequencing analysis of the potatoes’ inter-root soil microorganisms under the different fertilization treatments was carried out using ITS amplicon sequencing technology. In this experiment, six treatments with three replications were used for a total of 18 inter-root soil samples. The sequencing yielded 1,043,514 valid sequences, with an average length of 241 bp, and a total of 9149 OTUs were obtained from these sequences, with a sequence similarity level of 97%. The high-throughput sequencing technology was used to classify the 18 after sequencing and species annotation classifications of the fungal ITS regions in the test soil samples (OTUs after leveling by the minimum number of sample sequences); 1, 10, 34, 79, 183, 386, and 643 were obtained for the kingdom, phylum, order, family, genus, and species, respectively. Plotting the sample coverage curve of each treatment (Figure 1) shows that the index of each treatment group in the graph is greater than 0.99, converging to 1. It indicates that the sample size of this sequencing meets the requirement of precision, and the data quality is reliable and meets the requirements of experimental analysis.

3.3. Analysis of Alpha Diversity Index of Potatoes’ Inter-Root Soil Fungi Under Organic Fertilizer Replacing Nitrogen Fertilizer

The alpha diversity of the inter-root soil fungi of the potatoes with organic fertilizer replacing nitrogen fertilizer was analyzed, and the Chao and Shannon plots (Figure 2 and Figure 3) show that there is a decreasing trend of fungal richness in each treatment group on the Chao index compared with the CF treatment group, but there is no significant difference. This indicates that organic fertilizer replacing nitrogen fertilizer has no significant effect on the abundance of the inter-root soil fungi of potatoes. The Shannon index of each treatment group compared with the CF treatment group has a significant difference (p < 0.05), of which the T2 treatment group is significantly reduced by 7.85%. This indicates that organic fertilizer replacing nitrogen fertilizer can significantly reduce the diversity of potato inter-root soil fungi.

3.4. An Analysis of the Composition of the Inter-Root Soil Fungal Community of Potato Plants Under the Application of Organic Fertilizers in Place of Nitrogen Fertilizers

From the after sequencing and species annotation classification of the fungal ITS regions in the 18 test soil samples using high-throughput sequencing technology, a total of 10 species of fungal phyla were detected in the soil samples, as shown in the relative abundance plot of phyla-level community composition (Figure 4), of which the dominant phylum, with a relative abundance of more than 10%, was Ascomycota, which accounted for 85.84% of the overall community of fungi. The dominant phyla with a relative abundance of less than 10% included Mortierellomycota, Basidiomycota, Chytridiomycota, etc. These phyla accounted for 85.84% of the overall fungal community. They accounted for 6.91%, 3.69%, and 1.50% of the overall fungal community, respectively; in addition, there were fungal sequences that could not be annotated to a specific phylum, accounting for 1.93% of the overall fungal community, which indicates that there are still a certain number of fungal taxa in the soil that cannot be recognized at the level of the phylum.

The abundance of Ascomycota and Basidiomycota tended to increase in each treatment group compared with the CF treatment group. Ascomycota had the largest increase of 5.59% in the T2 treatment group, and Basidiomycota had the largest increase of 7.93% in the T3 treatment group. However, none of these reached a significant level.

From the after sequencing and species annotation classification of the fungal ITS regions in the 18 test soil samples using high-throughput sequencing technology, there were a total of 386 species of fungi at the genus level in the soil samples, and the relative abundance map of the genus-level community composition (Figure 5) is shown. The top 10 dominant species at the genus level, in terms of abundance, are listed for analysis, and among them, the species with a relative abundance of more than 10% is Chaetomium, which accounts for 10.75% of the overall fungal community. The dominant phyla with a relative abundance of less than 10% are Mortierella, Plectosphaerella, Trichocladium, Fusarium, Pseudombrophila, Lectera, Acremonium, and Penicillium, at 6.89%, 6.87%, 6.73%, 5.80%, 2.67%, 2.58%, 2.21%, and 2.18% of the overall fungal community, respectively. In addition, there are also fungal sequences that cannot be annotated to specific genera, accounting for 17.26% of the overall fungal community, indicating that there are still a certain number of fungal taxa in the soil that cannot be recognized at the genus level.

Among them, the abundance of Mortierella is significantly different (p < 0.05) from the CF treatment group, with a significant decrease of 46.45% in the T4 treatment group. The abundance of Chaetomium, Fusarium, and Plectosphaerella in each of the treatment groups show an increase in abundance compared to the CF treatment group. Chaetomium has the largest increase of 117.33% in the T3 treatment group; Fusarium has the largest increase of 105.15% in the T2 treatment group; Plectosphaerella has the largest increase of 44.03% in the T2 treatment group; and Trichocladium tends to decrease in abundance in all the treatment groups compared to the CF treatment group, and has the largest decrease of 58.28% in the T4 treatment group, but none of these reach significant levels.

3.5. Beta Diversity Analysis of Potatoes’ Inter-Root Soil Fungal Communities Under Organic Fertilizer Replacing Nitrogen Fertilizer

The Beta diversity can reflect the fungal diversity differences among samples. The results of the principal coordinate analysis of the inter-root soil fungal communities of the potatoes under the different fertilization treatments are shown in (Figure 6), and the contribution rates of the PC1 and PC2 axes are 24.84% and 11.79%, respectively. Along the PC1 axis, the CK treatment group is on the negative axis, and the other treatment groups gradually increase with the replacement rate of organic fertilizer, and each treatment group gradually extends further on the positive axis from the negative axis. Along the PC2 axis, all the treatment groups are distributed on both sides of the PC2 axis. Overall, the CK treatment groups are clustered together individually; the CF and T1 treatment groups are clustered together; and the T2, T3, and T4 treatment groups are clustered together; indicating that the similarity of the structural composition of the soil fungal community is higher for the 25% and 50% substitution rates, and the similarity of the structural composition of the soil fungal community is higher for the 50%, 75%, and 100% substitution rates. There are significant differences (p < 0.05) in the fungal community structure composition among the treatment groups, indicating that organic fertilizer replacing nitrogen fertilizer significantly changes the inter-root soil fungal community structure composition of potatoes.

3.6. Differential Analysis of Inter-Root Soil Fungal Communities of Potatoes Under Different Amounts of Organic Fertilizer Replacing Nitrogen Fertilizer

In order to gain a deeper understanding of the effects of fertilizer treatments on the inter-root fungal community, and to clarify the differential microorganisms among the treatments, an LEfSe analysis was performed to remove the unclassified microorganisms from the inter-root soil fungi of the potatoes under different amounts of organic fertilizers replacing nitrogen fertilizers, as shown in (Figure 7). On this basis, a histogram of the distribution of LDA values was drawn (Figure 8), and the results show that the diversity of the potatoes’ inter-root soil community is related to the different amounts of organic fertilizer replacing nitrogen fertilizer. The differential fungi in the CK treatment group were mainly Bradymyces_graniticola, Bradymyces, Trichomeriaceae, etc.; the differential fungi in the CF treatment group were mainly Mortierella_alpina, Aphanoascus, etc.; there were no obvious differential fungi in the T1 treatment group; the different fungi in the T2 treatment group were mainly Hannaella_luteola; the different fungi in the T3 treatment group were mainly Chaetomium, Penicillium, Chrysosporium, etc.; the different fungi in the T4 treatment group were mainly Acaulium, Aphanoascus, and so on. The differential fungi in each treatment group increased gradually with an increase in the organic fertilizer substitution rate. Among them, the T1 treatment had the lowest number of differential fungi and the T4 treatment group had the highest number of differential fungi. This indicates that organic fertilizer replacing nitrogen fertilizer changes the community structure of the fungi in potato inter-root soil and increases the differential fungi in soil.

3.7. Predictive Analysis of Inter-Root Soil Fungal Community Function in Potatoes Under Organic Fertilizer Replacing Nitrogen Fertilizer

FUNGuild (Fungi Functional Guild) is commonly used for functional analyses of fungal communities, and can distinguish between species that utilize similar environmental resources through similar pathways. Depending on the mode of nutrition, fungi can be categorized into Pathotrophs, Saprotrophs, and Symbiotrophs. The inter-root soil fungal communities of potatoes with different organic fertilizers replacing nitrogen fertilizers can be classified according to the mode of resource utilization, as Pathotroph, Symbiotroph, Saprotroph, Pathotroph–Saprotroph–Symbiotroph, Pathotroph–Symbiotroph, Pathotroph–Saprotroph, Saprotroph–Symbiotroph, and Pathotroph–Saprotroph–Saprotroph trophic types, as well as unspecified species of fungi. The highest percentage of the species were unclassified fungal species at 43.62%, followed by Saprotroph and Pathotroph–Saprotroph–Symbiotroph trophic types at 27.60% and 10.39%, respectively. The rest of the trophic types had a lower percentage. The application of organic fertilizers reduced the abundance of soil Pathogenic fungi and increased the abundance of Saprophytic fungi to varying degrees, and there was a tendency for the functional community of fungi to shift from Pathotrophic to Saprotrophic, which is conducive to the sustainable development of soil ecosystems.

In order to investigate whether there were differences in the function of the fungal types in the inter-root soil of the potatoes under different amounts of organic fertilizer replacing nitrogen fertilizer treatments, the top 10 categories of fungal ecological functions in the soil were selected for the statistical function prediction diagram shown in Figure 9. Among them, undefined Saprotroph, unknown species, Plant Pathogen, Endophyte–Litter Saprotroph–Soil Saprotroph–Undefined Saprotroph, Animal Pathogen–Dung Saprotroph–Endophyte–Epiphyte–Plant Saprotroph–Wood Saprotroph, Animal Pathogen-Endophyte-Lichen Parasite-Plant Pathogen- Soil Saprotroph-Wood Saprotroph, whose percentages were 23.58%, 12.97%, 13.67%, 6.89%, 11.92%, and 5.80%, respectively, were the major fungal functions in the soil.

Among them, unknown species and Endophyte–Litter Saprotroph–Soil Saprotroph–Undefined Saprotroph fungal functions showed a decreasing trend in each treatment group compared to the CF treatment group. Undefined Saprotroph and Plant Pathogen fungal functions tended to increase and decrease in each treatment group compared to the CF treatment group. Animal Pathogen–Dung Saprotroph–Endophyte–Epiphyte–Plant Saprotroph–Wood Saprotroph and Animal Pathogen–Endophyte–Lichen Parasite–Plant Pathogen–Soil Saprotroph–Wood Saprotroph fungal functions showed an increasing trend in each treatment group compared to the CF treatment group, but none of them reached a significant level of difference. With an increase in the organic fertilizer substitution rate, the unknown function of fungi and the single function of fungi appeared to reduce the trend of functional diversity; the complex fungi gradually increased, and the function of the fungal community gradually moved from a single to a diversified, complex evolution.

3.8. Correlation Analysis Between Inter-Root Soil Fungal Communities and Soil Environmental Factors of Potatoes Under Organic Fertilizer Replacing Nitrogen Fertilizer

To further reveal the main environmental factors affecting soil fungal communities, correlation analyses were conducted between the composition of soil fungal communities and soil environmental factors in the different treatments at the phylum level, plotting the correlation and RDA (Figure 10 and Figure 11). Mortierellomycota showed a highly significant positive correlation with soil pH (p < 0.01), a significant negative correlation with total nitrogen (p < 0.05), a very highly significant negative correlation with quick-acting phosphorus (p < 0.001), a highly significant negative correlation with alkaline dissolved nitrogen (p < 0.01), and a highly significant negative correlation with quick-acting potassium (p < 0.01). Chytridiomycota was significantly and positively correlated (p < 0.05) with alkaline dissolved nitrogen. There was no significant correlation between the total potassium, total phosphorus, organic matter, and soil fungal community phylum levels. From the RDA plot, it can be seen that soil pH is the main environmental factor for the inter-root soil portal level of the fungal community in potatoes with organic fertilizer replacing nitrogen fertilizer.

A correlation analysis was conducted between the soil fungal community composition and the soil physicochemical factors of the different treatments at the genus level, and we plotted the correlation with RDA (Figure 12 and Figure 13). Chaetomium showed a very highly significant positive correlation with total potassium (p < 0.001), a highly significant positive correlation with quick-acting phosphorus and organic matter (p < 0.01), a significant negative correlation with alkaline dissolved nitrogen and quick-acting potassium (p < 0.05), and a highly significant negative correlation with pH (p < 0.01). Mortierella showed a highly significant positive correlation (p < 0.01) with pH, a very highly significant negative correlation (p < 0.001) with quick-acting phosphorus, a highly significant negative correlation (p < 0.01) with alkaline dissolved nitrogen and quick-acting potassium, and a significant negative correlation (p < 0.05) with total nitrogen. Trichocladium showed a very highly significant positive correlation with pH (p < 0.001), a highly significant negative correlation with total nitrogen and quick-acting potassium (p < 0.01), and a significant negative correlation with alkaline dissolved nitrogen (p < 0.05). Fusarium showed a very highly significant negative correlation (p < 0.001) with pH, highly significant positive correlations (p < 0.01) with total nitrogen and quick-acting potassium, and significant positive correlations (p < 0.05) with total phosphorus and alkaline dissolved nitrogen. unclassified_p_Ascomycota showed a significant positive correlation (p < 0.05) with pH and a significant negative correlation (p < 0.05) with total nitrogen, total potassium, and quick-acting potassium. From the RDA plot, it can be seen that soil pH is the main environmental factor affecting the fungal community at the genus level in the inter-root soil of potatoes with organic fertilizer replacing nitrogen fertilizer.

4. Discussion

Soil microorganisms play a crucial role in the formation and cycling of soil organic carbon and the release of soil nutrients. Fungi are an integral part of the soil microbial community and are closely linked to soilborne diseases and plant interactions [29]. The application of organic fertilizers significantly increased the content of alkaline dissolved nitrogen, quick-acting potassium, quick-acting phosphorus, and organic matter in the inter-root soils of the potatoes, and significantly lowered the pH of the soil. However, the effect on the soils’ total nitrogen, total potassium, and total phosphorus was not significant. These results are in agreement with the results of Assefa [30] and Lacolla [31], who studied the ability of organic fertilizers to reduce soil pH and change a soil’s physicochemical properties. They showed a significant increase in the quick-acting nutrients in the soil with an increase in the organic fertilizer substitution rate, but the differences between the treatments for elements such as total phosphorus were not significant. This may be due to the fact that organic matter increases microbial activity, which allows for the conversion of the total nitrogen, phosphorus, and potassium in soil into quick-acting nutrients, resulting in an increase in quick-acting nutrients. The input of phosphorus into the soil is basically the same and can meet the growth requirements of potatoes, so there is no significant difference in the whole element between the organic replacement treatment groups. Through the study of the Alpha diversity index, we found that the number of fungal species in the potatoes’ inter-root soil under organic fertilizer replacing nitrogen fertilizer had a tendency to decrease in each treatment group compared to the CF treatment group, which did not reach the level of significance. This indicates that organic fertilizer replacing nitrogen fertilizer had no significant effect on the abundance of the inter-root soil fungi of the potatoes, and the number of fungal species in each treatment group was significantly different (p < 0.05) compared with the CF treatment group, and was significantly reduced by 7.85% in the T2 treatment group. This indicates that organic fertilizer replacing nitrogen fertilizer significantly reduced the diversity of the inter-root soil fungi of the potatoes. This result is consistent with the findings of Yu Weilong [32] et al., who found that with the application of an organic fertilizer treatment the fungal Shannon index significantly decreased and the fungal diversity decreased, and the application of an organic fertilizer treatment significantly decreased the Chao index and fungal abundance compared to a CF treatment. This result is consistent with Peng [33] and Lee [34], who argued that the application of an organic fertilizer is an important measure for soil improvement and increasing soil fertility. Organic fertilizer can effectively change the species and number of soil microorganisms, thus affecting the structure and spatial distribution structure of a soil bacterial community. This result is inconsistent with the results of Yang [35] et al. It may be that the application of organic fertilizers reduces the soil pH, which is close to neutral, affecting the structure of fungal communities, reducing the number of fungi adapted to alkaline environments, and decreasing diversity. Organic fertilizers increase organic matter, provide carbon and energy sources, change the competition and interactions among fungi, and affect soil environmental factors, which in turn affect fungal diversity. The geographic environment, farming methods, fertilizer application, and fertilization methods had a greater impact on the diversity of fungi in the soil, due to the more obvious changes in rainfall in the test area during the experimental year. During the period of low rainfall, the potato expansion period caused by soil microbial activity was weaker. We found that organic fertilizer replacing nitrogen fertilizer had a significant effect on the potatoes’ inter-root soil fungal community with respect to β-diversity. A principal coordinate analysis of the fungal community composition of the potatoes’ inter-root soils revealed that the structural composition of the fungal community in each treatment group could be classified into three major categories with an increasing organic fertilizer substitution rate, with the CK treatment group as the first category; the CF and T1 treatment groups as the second category; and the T2, T3, and T4 treatment groups as the third category. This indicates that organic fertilizer replacing nitrogen fertilizer significantly changed the inter-root soil fungal community structure of the potatoes. The LEfSe analysis of the soil fungi revealed that the differential fungi in each treatment group gradually increased with an increase in the organic fertilizer replacement rate. The T1 treatment had the lowest number of differential fungi and the T4 treatment group had the highest number of differential fungi. This indicates that organic fertilizers replacing nitrogen fertilizers changed the community structure of the fungi in the potatoes’ inter-root soil and increased the differential fungi in the soil. Among the differential fungi, those involved in organic matter decomposition, organic matter cycling, and plant interactions accounted for the largest proportion. It is possible that the addition of organic matter to the soil resulted in changes in the soil nutrient dynamics; with a significant increase in the fast-acting nutrients, there was an increase in the number of fungal species favorably involved in organic matter decomposition and cycling in the soil, and an increase in the number of species of fungi adapted to the dynamic nutrient changes due to the organic matter, which further impacted the variability in the fungal species among the treatments. Through the soil fungi FUNGuild function prediction analysis study, we found that with an increase in the organic fertilizer substitution rate, the unknown function of fungi was significant and the single function of fungi appeared to reduce the trend of functional diversity, and the complexity of the fungi gradually increased; the fungal community function gradually transformed from single to diverse and complex. This result is in line with that of Yang [36], who found that soil fertility and plant growth are inextricably linked to the soil microbial communities. The improved microbial community diversity results are consistent. It may be that the distribution of organic fertilizers can significantly increase the relative abundance of Saprophytic fungi, and the main functions of Saprophytic nutrient fungi are to decompose organic matter, promote nutrient cycling, improve soil structure and fertility, participate in carbon cycling, inhibit the growth of pathogenic bacteria, and promote plant growth. This indicates that the function of fungi in the soil is mainly to decompose organic matter, promote nutrient cycling, and promote nitrogen cycling, and to improve the structure and fertility of the soil due to the addition of organic matter to the soil. Organic fertilizers promote the growth of the number and species of Saprophytic fungi with the function of decomposing organic matter, which is favorable to the growth and development of potatoes. Through the correlation analysis of environmental factors, it can be seen that the soil pH, alkaline dissolved nitrogen, quick-acting potassium, quick-acting phosphorus, and organic matter are environmental factors affecting the inter-root soil fungal community of potatoes with organic fertilizer replacing nitrogen fertilizer. Among them, the soil pH is the main environmental factor affecting the inter-root soil fungal communities of potatoes with organic fertilizer replacing nitrogen fertilizer. The continuous application of organic fertilizers helps to regulate the pH of saline soils towards neutrality, improve soil structure, and enhance soil fertility. This process enhances the effectiveness of nutrients. For example, it reduces the binding of phosphorus to calcium and increases the availability of phosphorus. At the same time, lowering the pH increases calcium ions, promotes soil structure formation, enhances soil fertility, and activates soil microbes for nutrient conversion. In addition, an appropriate pH range (6.5 to 7.5) favors crop growth and reduces salts in the soil that are harmful to crop growth.

At the phylum level, Ascomycota, Mortierellomycota, Basidiomycota, and Chytridiomycota were the major core flora. In this study, it was found that there was a significant difference (p < 0.05) in the amount of Mortierellomycota in each treatment group as compared to the CF treatment group, and in the T4 treatment group it was significantly reduced by 46.62%. This result is consistent with the finding of Wei Quanquan [37] that the relative abundance of Mortierellomycota in the soil is reduced by nitrogen fertilizer and organic materials. Mortierellomycota has the ability to degrade agricultural wastes and organic pollutants to repair the soil, dissolve phosphorus and iron production carriers to promote the transformation of soil nutrients, secrete phytohormones and fatty acid analogs to promote plant growth, secrete antagonistic substances to regulate the abundance of inter-root microbial populations, and induce a plant’s defense response to improve plant disease resistance [38]. From the results of our correlation analysis of environmental factors, although the addition of organic fertilizers lowered the soil pH and promoted the growth of Mortierellomycota, the increase in fast-acting phosphorus, alkaline dissolved nitrogen, and fast-acting potassium, in turn, inhibited the growth of Mortierellomycota, which ultimately led to a decrease in the number of Mortierellomycota. Basidiomycota tended to decrease in abundance in each treatment group compared to the CF treatment group. Basidiomycota is able to efficiently decompose lignified plant debris and possesses an excellent ability to break down lignocellulose in plant residues [39]. The decrease in its relative content may hinder the decomposition process of lignocellulose in plant debris. Ascomycota tended to increase in abundance in each treatment group compared with the CF treatment group. Ascomycota are mainly Saprophytic bacteria that survive in the soil in a multitude of nutrient ways and can break down organic matter in the soil into nutrients that can be absorbed by crops, while increasing the organic matter content of the soil, thus providing nutrients that are available to crops [40]. The increase in Ascomycota is conducive to the enrichment of the decomposition capacity of soil fungal microorganisms, which contributes to the improvement of the soil’s ecological environment.

At the genus levels, Chaetomium, Mortierella, Plectosphaerella, Trichocladium, and Fusarium were the major core flora. In the present study, a significant difference (p < 0.05) was found in the number of Mortierella spp. in all treatment groups as compared to the CF treatment group, with a significant reduction of 46.45% in the T4 treatment group. Mortierella spp. decompose phosphorus, cellulose, and hemicellulose in the soil, increase soil nutrients, and have an antagonistic effect on phytopathogenic fungi [41]. The reasons for the changes in the number of Mortierella fungi are consistent with the reasons for Mortierellomycota mentioned above. The abundance of Chaetomium, Fusarium, and Plectosphaerella tended to increase in each treatment group compared to the CF treatment group. Chaetomium is effective for the degradation of cellulose and organic matter, and has an antagonistic effect on other microbial organisms in soil [42]. From the correlation analysis of environmental factors, although the addition of organic fertilizer lowered the soil pH and inhibited the growth of Chaetomium, the increase in fast-acting potassium promoted the growth of Chaetomium, which ultimately led to an increase in the number of Chaetomium. Fusarium and Plectosphaerella are the main pathogenic fungi that cause destructive diseases, and their effects on the inter-root zone of potatoes are mainly seen in causing potato dry rot, which is a serious soil-borne disease that leads to a decrease in potato yield [43]. The application of nitrogen fertilizer can increase the content of ammonium nitrogen in soil. The cumulative release of ammonium nitrogen in the soil by mixing nitrogen fertilizer with organic fertilizer, and the application of organic fertilizer alone, can increase the utilization of ammonium nitrogen and reduce the loss of ammonium nitrogen [44]. Pathogenic bacteria under ammonium nitrogen supply conditions can effectively utilize carbon sources in the soil to occupy ecological niches and promote spore germination and toxin production by further altering the metabolism of sugars and amino acids in a plant, which ultimately leads to a significant increase in the number of pathogenic bacteria [45]. From the results of the correlation analysis of environmental factors, although the addition of organic fertilizer reduced the pH of the soil and inhibited the growth of pathogenic bacteria, the increase in organic matter, alkaline dissolved nitrogen, and quick-acting potassium, in turn, promoted the growth of pathogenic bacteria, which ultimately led to an increase in the number of pathogenic bacteria. This result is consistent with the results of Ma [46], who found Fusarium to be the dominant population in organic fertilizer treatments.

The main production areas of potatoes have had a long period of continuous cropping, and continuous cropping also changes the structural composition of the fungal community to a certain extent. Continuous cropping leads to an increase in the number of disease-causing bacteria in the soil and an increase in the risk of potato plants being infected with soil-borne diseases, especially increasing the chances of blight in potatoes. It is possible that the increase in the number of Fusarium spp. in this paper is due to continuous potato cultivation. Due to the high soil pH in the main potato-producing areas of Gansu, the soil is weakly alkaline, and may appear to inhibit certain fungi, and the addition of organic fertilizers significantly reduces the soil pH value. However, the correlation analysis of the environmental factors showed that the decrease in pH had highly significant effects on many fungal populations, followed by highly significant effects on nutrient factors, such as alkaline dissolved nitrogen and fast-acting potassium. This indicates that soil fungi are more sensitive to pH. Further studies are needed to address the changes in soil fungal communities for different organic fertilizer replacement ratios and years of continuous cropping.

5. Conclusions

The organic fertilizer replacing some of the chemical fertilizers significantly increased the content of alkali dissolved nitrogen, quick-acting potassium, quick-acting phosphorus, and organic matter in the potatoes’ inter-root soil, and significantly reduced the pH value of the soil, while the total nitrogen, phosphorus, and potassium in the soil tended to stabilize, and the differences were not significant. The organic fertilizers replacing some of the chemical fertilizers had a tendency to reduce the abundance of soil fungi and could significantly reduce the α-diversity of soil fungi. The core fungi in the soil in each treatment group were Acomycota, Mortierellomycota, Basidiomycota, etc. The replacement of nitrogen fertilizer by organic fertilizer significantly altered the structural composition of the inter-root soil fungal communities of the potatoes and increased the differential fungi in the soil. The fungal community function gradually shifted from Singularity to diversification and complexity. The redundancy analysis showed that the soil pH was the main environmental factor affecting the inter-root soil fungal communities of potatoes under organic fertilizer replacing N fertilizer. The application of organic fertilizers reduced soil pathogen abundance and increased Saprophytic fungal abundance to varying degrees, with a tendency for the functional community of fungi to shift from Pathotrophic to Saprotrophic.

It is recommended that organic fertilizers be used in conjunction with chemical fertilizers to reduce nitrogen fertilizer losses, improve utilization efficiency, and reduce agricultural surface pollution. The results of this study showed that in the potato-growing area of Gansu in the Loess Plateau region, 50% organic fertilizer replacing nitrogen fertilizer is the optimal ratio for potato cultivation.