Influence of Fungicide Application on Rhizosphere Microbiota Structure and Microbial Secreted Enzymes in Diverse Cannabinoid-Rich Hemp Cultivars

Abstract

Microbes and enzymes play essential roles in soil and plant rhizosphere ecosystem functioning. However, fungicides and plant root secretions may impact the diversity and abundance of microbiota structure and enzymatic activities in the plant rhizosphere. In this study, we analyzed soil samples from the rhizosphere of four cannabinoid-rich hemp (Cannabis sativa) cultivars (Otto II, BaOx, Cherry Citrus, and Wife) subjected to three different treatments (natural infection, fungal inoculation, and fungicide treatment). DNA was extracted from the soil samples, 16S rDNA was sequenced, and data were analyzed for diversity and abundance among different fungicide treatments and hemp cultivars. Fungicide treatment significantly impacted the diversity and abundance of the hemp rhizosphere microbiota structure, and it substantially increased the abundance of the phyla Archaea and Rokubacteria. However, the abundances of the phyla Pseudomonadota and Gemmatimonadetes were substantially decreased in treatments with fungicides compared to those without fungicides in the four hemp cultivars. In addition, the diversity and abundance of the rhizosphere microbiota structure were influenced by hemp cultivars. The influence of Cherry Citrus on the diversity and abundance of the hemp rhizosphere microbiota structure was less compared to the other three hemp cultivars (Otto II, BaOx, and Wife). Moreover, fungicide treatment affected enzymatic activities in the hemp rhizosphere. The application of fungicides significantly decreased enzyme abundance in the rhizosphere of all four hemp cultivars. Enzymes such as dehydrogenase, dioxygenase, hydrolase, transferase, oxidase, carboxylase, and peptidase significantly decreased in all the four hemp rhizosphere treated with fungicides compared to those not treated. These enzymes may be involved in the function of metabolizing organic matter and degrading xenobiotics. The ecological significance of these findings lies in the recognition that fungicides impact enzymes, microbiota structure, and the overall ecosystem within the hemp rhizosphere.

Keywords: Cannabis sativa; abundance; cannabinoid (CBD); diversity; enzymes; fungicide; hemp rhizosphere.

Figure 1

The relative abundance of the top 10 (out of 31) phyla of bacteria on the hemp rhizosphere at different treatments. Note: Otto II, BaOx, Wife, Cherry Citrus with fungicide treatment are denoted by D, K, G, L, respectively. Otto II, BaOx, Wife, Cherry Citrus with natural infection are represented by F, E, O, J, and Otto II, BaOx, Wife, Cherry Citrus with fungal inoculation are indicated by A, C, I, H.

Figure 2

Principal component analysis (PCA) of the relative abundance of the 42 samples across the 3 fungicide treatments and 4 hemp cultivars. Note: Otto II, BaOx, Wife, Cherry Citrus with fungicide treatment are denoted by D, K, G, L (in the green circle), respectively. Otto II, BaOx, Wife, Cherry Citrus with natural infection are represented by F, E, O, J (in the purple circle), and Otto II, BaOx, Wife, Cherry Citrus with fungal inoculation are indicated by A, C, I, H (in the red circle).

Figure 3

Non-metric multidimensional scaling (NMDS) of the relative abundance of the 42 samples across the 3 fungicide treatments and 4 hemp cultivars. Note: Otto II, BaOx, Wife, Cherry Citrus with fungicide treatment are denoted by D, K, G, L (in the green circle with pentagon symbols), respectively. Otto II, BaOx, Wife, Cherry Citrus with natural infection are represented by F, E, O, J (in the purple circle with downward triangle symbols), and Otto II, BaOx, Wife, Cherry Citrus with fungal inoculation are indicated by A, C, I, H (in the red circle with upward triangle symbols).

Figure 4

The relative abundance (RA) of the top 12 (out of 31) phyla between fungicide-treated and other treatments in the hemp rhizosphere. Note: The ratios of fungicide-treated to fungal inoculation for the hemp cultivars Otto II, BaOx, Cherry Citrus, Wife are represented by A.D, C.K, H.L, I.G, while the ratios of fungicide-treated to natural infection for the same cultivars are denoted by F.D, E.K, J.L, O.G. Two color scales are utilized, with blue indicating low abundance and red indicating high abundance in fungicide-treated samples compared to fungal inoculation or natural infection.

Figure 5

The relative abundance of bacteria that significantly varies among three distinct treatments in the rhizosphere of four hemp cultivars. The treatments include fungal inoculation (A, C, I, H), natural infection (F, E, O, J), and fungicide treatment (D, K, G, L) across the hemp cultivars Otto II, BaOx, Wife, and Cherry Citrus. The vertical axis represents the relative abundance [Log2 (relative abundance +1)] of each bacterial phylum, while the horizontal axis displays all microbial phyla that exhibit significant differences across the three treatments within the rhizosphere (P < 0.05). The four hemp cultivars are clearly labeled for easy identification. Overall, this figure illustrates the dynamic microbial composition within hemp plant rhizospheres under varying treatment conditions, emphasizing the specific bacterial phyla that significantly respond to these treatments across the four cultivars.

Figure 6

The relative abundance of bacteria that significantly varies across four hemp cultivars in the rhizosphere under three treatments. These cultivars are Otto II, BaOx, Wife, and Cherry Citrus, subjected to fungal inoculation (A, C, H, I), fungicide treatment (D, K, L, G), and natural infection (F, E, J, O). The vertical axis displays the relative abundance [Log2 (relative abundance +1)] of each bacterial phylum, while the horizontal axis lists the microbial phyla with significant differences among the cultivars under the three treatments (P < 0.05). The legend clarifies the three treatments (fungal inoculation, fungicide treatment, and natural infection). This figure provides insights into distinct bacterial populations in the rhizosphere of different hemp cultivars under varied treatment conditions, highlighting significant variations in bacterial abundance among cultivars across the treatments.

Figure 7

The relative abundance of Clusters of Orthologous Genes (COGs) and a Venn diagram highlighting shared COGs that show significant differences between fungicide-treated and non-fungicide-treated for the four hemp cultivars. (A): This panel displays the number of COGs that are significantly down-regulated (down) and up-regulated (up) (P < 0.05) or non-significantly regulated (nonSig) (P < 0.05) between fungicide-treated versus fungal inoculation, or fungicide-treated versus natural infection, across the four hemp cultivars (e.g., Otto II, BaOx, Wife, and Cherry Citrus). (B) shows the shared COGs significantly down-regulated in fungicide treatment compared to natural infection across the four hemp cultivars. (C) depicts the shared COGs significantly down-regulated in fungicide treatment compared to fungal inoculation for the four hemp cultivars. (D) highlights the shared COGs significantly up-regulated in fungicide treatment compared to natural infection for the four hemp cultivars. (E) presents the shared COGs significantly up-regulated in fungicide treatment compared to fungal inoculation for the four hemp cultivars. These insights offer a detailed view of how fungicide treatment influences the regulation of COGs within different hemp cultivars, shedding light on specific genetic responses to fungicide exposure.

Figure 8

The relative abundance of enzymes and a Venn diagram illustrating shared enzymes that exhibit significant differences between fungicide-treated and non-fungicide-treated hemp cultivars. (A): This panel quantifies the number of enzymes that are significantly down-regulated (down) and up-regulated (up) (P < 0.05) or non-significantly regulated (nonSig) (P < 0.05) between fungicide-treated versus fungal inoculation, or fungicide-treated versus natural infection, across the four hemp cultivars (e.g., Otto II, BaOx, Wife, and Cherry Citrus). (B) demonstrates the shared enzymes significantly down-regulated in fungicide treatment compared to natural infection across the four hemp cultivars. (C) illustrates the shared enzymes significantly down-regulated in fungicide treatment versus fungal inoculation for the four hemp cultivars. (D) highlights the shared enzymes significantly up-regulated in fungicide treatment compared to natural infection for the four hemp cultivars. (E) presents the shared enzymes significantly up-regulated in fungicide treatment compared to fungal inoculation for the four hemp cultivars. These insights offer a detailed look at how fungicide treatment affects enzyme regulation in diverse cannabinoid-rich hemp cultivars, revealing specific enzyme responses to fungicide exposure.

Figure 9

The relative abundance of pathways and a Venn diagram depicting shared pathways that display significant differences between fungicide-treated and non-fungicide-treated hemp cultivars. (A): This panel quantifies the number of pathways significantly down-regulated (down) and up-regulated (up) (P < 0.05) or non-significantly regulated (nonSig) (P < 0.05) between fungicide-treated versus fungal inoculation, or fungicide-treated versus natural infection, across the four hemp cultivars (e.g., Otto II, BaOx, Wife, and Cherry Citrus). (B) demonstrates the shared pathways significantly down-regulated in fungicide treatment compared to natural infection across the four hemp cultivars. (C) illustrates the shared pathways significantly down-regulated in fungicide treatment compared to fungal inoculation for the four hemp cultivars. (D) highlights the shared pathways significantly up-regulated in fungicide treatment compared to natural infection for the four hemp cultivars. (E) presents the shared pathways significantly up-regulated in fungicide treatment compared to fungal inoculation for the four hemp cultivars. These insights provide a thorough understanding of how fungicide treatment affects the regulation of pathways in various hemp cultivars, offering specific insights into how pathways respond to fungicide exposure. The relative abundance of pathways and the Venn diagram of shared pathways significantly differed between fungicide-treated and non-fungicide-treated in the different hemp cultivars.

Figure 10

The experimental layout of four hemp cultivars within the field with each subjected to different treatments. Note that the four distinct hemp cultivars—Otto II, BaOx, Wife, and Cherry Citrus—are allocated to individual plots. Within each plot, three distinct treatments are applied to the respective hemp cultivars: fungicide treatment, fungal inoculation, and natural infection. This systematic and methodical design employs a randomized layout to mitigate biases and enhance the reliability of statistical analyses.