3.1 Field investigation on main diseases of ginseng planting under forest in different treatments
The seedling survival rate of mountain-cultivated ginseng was significantly increased in rain-shelter cultivation (Fig. 2A, 2B). The incidence of gray mold and black spot diseases was highest during the red fruit stage, while the disease index was highest during the flowering stage. In rain-shelter cultivation, the incidence rates of gray mold in the four periods, respectively, were 7.43%, 8.12%, 10.33%, and 8.11%, and 10.22%, 16.13%, 19.51%, and 12.15% of black spot. The disease index of gray mold was 5.12, 6.88, 6.73, and 5.97, and it was 5.21, 8.87, 7.82, and 6.33 for black spot. In open-field cultivation, the incidence rates of gray mold in the four periods, respectively, were 6.98%, 7.21%, 11.2%, and 7.56%, and 8.77%, 9.67%, 14.82%, and 13.21% of black spot. The disease index of gray mold was 5.27, 6.31, 5.11, and 4.98, and that of black spot was 6.58, 7.31, 6.33, and 5.21. (Fig. 2C). During the withering stage, we measured the incidence of root diseases, including rust rot and root rot. The incidence of rust rot in rain-shelter cultivation was 9.21%, while in open-field cultivation it was 15.48%. Similarly, the incidence of root rot in rain-shelter cultivation was 8.73%, while in open-field cultivation it was 14.12%. Field investigation showed that using rain-shelter cultivation resulted in a lower incidence and disease index compared to open field cultivation. (Fig. 2D).
3.2 Effects of rain-shelter cultivation on OTU and pathogenic fungi of ginseng rhizosphere soil
Fungal sequencing based on ITS rRNA genes used 97% nucleotide sequence identity. There were a total of 2373 OTUs in rain-shelter cultivation and open-field cultivation, 1365 unique OTUs of R and 1371 unique OTUs of O (Fig. 3). R1, R2, R3, and R3 have 1805, 1806, 1804, and 1638, respectively, among which there are 693, 439, 438, and 388 unique OTUs, respectively; R1 and R2 have 825 OTUs; R2 and R3 have 871 OTUs; and R3 and R4 have 853 OTUs. A total of 528 outs were shared across four samples. O1, O2, O3, and O4 have 1658, 2121, 1501, and 1493, respectively, among which there are 536,825,337,394 unique OTUs, respectively; O1 and O2 have 825 OTUs; O2 and O3 have 871 OTUs; and O3 and O4 have 853 OTUs. A total of 486 outs were shared across four samples. In addition, R1 and O1 have a total of 925 OTUs, R2 and O2 have a total of 970 OTUs, R3 and O3 have a total of 925 OTUs, and R4 and O4 have a total of 896 OTUs.
Ilyonectria, Cylindrocarpon, and Fusarium are the main pathogens of ginseng root diseases. According to Fig. 4, the quantity of Ilyonectria in open-field cultivation is 1.5 times higher than in rain-shelter cultivation throughout the year. Additionally, Fusarium levels initially increased and then decreased, rain-shelter cultivation consistently showed lower levels compared to open-field cultivation throughout the year. This difference was particularly significant during the flowering stage, with a 3.4 times lower incidence in rain-shelter cultivation. Botrytis and Alternaria are the main pathogens of ginseng leaf diseases. Similarly, during the growing season, the amount of Alternaria in open-field cultivation is 2 to 4 times greater than in rain-shelter cultivation. (Figure.4).
3.3 Effects of rain-shelter cultivation on the richness and diversity of ginseng rhizosphere soil community
In order to distinguish the fungal diversity of ginseng rhizosphere soil under a rain-shelter cultivation microenvironment, alpha diversity analysis was used to evaluate the two cultivation methods. In all collected samples, the estimated Good's coverage was close to 100%, indicating that most fungal taxa had been included.
Chao and Sobs were used to estimate the fungal richness index of the rhizosphere soil sample. According to the data, the fungal richness of rhizosphere soil is higher in rain-shelter cultivation than in open-field cultivation during each period (Table 2). Both the Simpson and Shannon indices are commonly used to estimate microbial diversity in a sample. The diversity gradually decreased in the rain-shelter cultivation mode. Although the budding stage was slightly higher than that of open-field cultivation, it was lower than that of open-field cultivation from the beginning of flowering (Table 2).
Table 2
Alpha diversity of fungi in samples
| coverage | chao | sobs | shannon | simpson |
R1 | 0.997 | 1036.81 ± 109.15 b | 982.0 ± 175.35 b | 5.00 ± 0.23 a | 0.020 ± 0.005 b |
O1 | 0.997 | 1038.22 ± 14.60 b | 967.0 ± 12.29 b | 4.89 ± 0.01 ab | 0.023 ± 0.001 b |
R2 | 0.996 | 1385.09 ± 68.92 a | 1269.7 ± 48.25 a | 4.58 ± 0.07 bc | 0.041 ± 0.004 a |
O2 | 0.995 | 1127.61 ± 148.16 ab | 1025.7 ± 110.96 b | 5.06 ± 0.05 a | 0.024 ± 0.003 b |
R3 | 0.996 | 1131.72 ± 125.82 ab | 1000.7 ± 92.41 b | 4.21 ± 0.14 de | 0.049 ± 0.006 a |
O3 | 0.996 | 992.98 ± 46.63 b | 856.7 ± 54.13 b | 4.51 ± 0.06 cd | 0.040 ± 0.004 a |
R4 | 0.996 | 1071.81 ± 39.10 b | 939.7 ± 28.5 b | 4.03 ± 0.11 e | 0.052 ± 0.001 a |
O4 | 0.996 | 956.61 ± 34.94 b | 837.0 ± 30.51 b | 4.09 ± 0.02 e | 0.051 ± 0.004 a |
3.4 Composition of rhizosphere soil fungi community in rain-shelter cultivation of ginseng
According to the annotation results of species sequences in the OTU of rhizosphere soil samples of each treatment, the top 15 species were selected to create a relative abundance histogram (Fig. 5A), which included Mortierella, Trichophaea, and Solicoccozyma. Among them, Mortierella showed a trend of increasing first and then decreasing in the rain-shelter cultivation mode. It increased from 19.21–31.01% from the budding stage to the red fruit stage but decreased to 25.16% in the withering stage. It was higher than the open-field cultivation from the flowering stage to the withering stage. The proportion of Trichophaea in each period under rain-shelter cultivation mode was 2.19%, 11.03%, 2.84%, and 13.56%, respectively, which was much higher than that of open-field cultivation. Cladosporium showed a trend of increasing first and then decreasing in rain-shelter cultivation, accounting for 1.85%, 2.03%, 0.47%, and 0.25%, respectively, in each period, which was lower than that in open-field cultivation from the red fruit stage. The proportion of Pseudeurotium in rain-shelter cultivation in each period was 2.58%, 0.46%, 1.02%, and 0.85%, respectively, and in open-field cultivation it was 1.99%, 0.57%, 3.07%, and 1.02%, respectively. On the annual scale, it showed a decreasing trend.
The Circos diagram is a visual representation of the relationship between samples and species. It reflects the proportion of dominant species in each sample and their distribution across different samples. More specifically, the main fungal genus identified in ginseng rhizosphere soil is Mortierella. The precipitation conditions decreased the colonization of Brachysteura and Clavosporium and increased the abundance of Mortierella and Trichodiscus, both belonging to ascomycetes. Other fungal phyla, including Mucor, basidiomycetes, and Rozobacteria, have also been detected. (Fig. 5B)
PCoA and NMDS analyses were conducted to compare species diversity between different habitats or microbial communities. The analyses reflected the degree of aggregation and dispersion of sample communities by the distance between samples and explored the similarity or difference in community composition between samples of different groups.
The PCoA analysis showed that the contribution rate of PCo1 was 46.84% and that of PCo2 was 26.38% (Fig. 6A). The clustering between rain-shelter cultivation and open-field cultivation in the budding stage was similar, and the distance between them widened during the subsequent growth of ginseng. It was further indicated that at the same growth stage, the community structure of fungi in rain-shelter cultivation was obviously different from that in open-field mode. The sampling time of R2 and R3 is in the rainy season, which is greatly affected by meteorology, but this effect is reduced under rain-shelter cultivation, so there is a certain intersection.
The results of non-metric multidimensional scaling analysis (NMDS) were similar to those of PCoA analysis. The clustering of rain-shelter cultivation and open-field cultivation in the budding stage of ginseng was similar. The distance between the two was continuously widened during the subsequent growth of ginseng, and there was an intersection between R2 and R3 (Fig. 6B). It was further verified that rain-shelter cultivation could affect the composition and structure of ginseng rhizosphere microorganisms.
ANOSIM/Adonis analysis was used to investigate the relationship between fungal community patterns and the growth stage of ginseng. A box plot was used to illustrate the differences between and within groups, highlighting the variations in the classification of detected fungi. The box-shaped distance between groups (Fig. 7A) shows that the differences between groups are much greater than the differences within groups. In addition, in the rhizosphere soil of rain-shelter cultivation and open-field cultivation modes (Bray-Curtis RANOSIM = 0.888, p < 0.001; R2 ADONIS = 0.773; 0.001), the fungal community structure was significantly different in the rhizosphere of ginseng in budding stage, flowering stage, red fruit stage, and withering stage. In rhizosphere soil, there were significant differences in fungal community structure between the two cultivation modes, which had significant effects on the taxonomic differences of fungi. Note The grouping is meaningful (Fig. 7B).
3.5 Correlation between microbial community diversity and meteorological factors
In this study, meteorological data such as air temperature, air humidity, soil temperature, and soil humidity were monitored through rain-shelter cultivation and open-field cultivation (Fig. 8).
The soil temperature of the rain-shelter cultivation was higher than that of the open-field cultivation after the flowering stage, and the temperature is always slightly higher than in open-field cultivation mode. Both air humidity and soil humidity exhibited an increasing trend from the budding stage to the red fruit stage, followed by a decreasing trend from the red fruit stage to the wither stage. The air humidity under rain-shelter cultivation and open-field cultivation can respectively reach 57.6% and 61.3%, and the soil humidity can respectively reach 61.3% and 78.1%. The humidity levels in the air and soil are typically lower in rain-shelter cultivation compared to open-field cultivation.
Redundancy analysis (RDA) was used to analyze the relationship between four growth periods (response variables) and four meteorological factors (explanatory variables) of mountain-cultivated ginseng. We observed that the structure and distribution of fungal microbiota in four growth periods were influenced by the modulation of all meteorological factors monitored. (Fig. 9). Upon further observation of the fungal community at the genus level, it was found that the soil microbial community was most affected by soil humidity, followed by soil temperature. This influence gradually increased over time. During the budding stage, the rain-shelter cultivation of ginseng was more affected by air temperature compared to open-field cultivation. This effect persisted until the flowering stage, possibly due to the altered microclimate caused by the rain-shelter cultivation, resulting in a higher air temperature.
Correlation Heatmap analysis involves calculating the correlation coefficient between environmental factors and selected species. The resulting numerical matrix is then visually displayed on a heatmap (Fig. 9B). The correlation heatmap analysis revealed that the abundance of Piloderma was positively correlated with soil moisture and air moisture and negatively correlated with soil temperature (***). Soil temperature (ST) significantly promoted the growth of Neonectria (***) and Mortierella (*). Soil moisture (SH) significantly inhibited the propagation of Solicoccozyma (*) and promoted the growth of Wilcoxina and Plectosphaerella (*). However, Wilcoxina is significantly inhibited by air temperature. Pseudeurotium fungi were also strongly inhibited by air temperatures (Fig. 9D).