Effects of a compound Trichoderma agent on Coptis chinensis growth, nutrients, enzyme activity, and microbial community of rhizosphere soil

Culture of Trichoderma

Trichoderma species: The Trichoderma atroviride, Trichoderma longibrachiatum, Trichoderma hamatum, and Trichoderma koningiopsis were all from the laboratory-preserved species.

Culture of Trichoderma strains: wheat grains were soaked in water for 20 h until swollen (humidity 45%) and drained. Then, in a 500-mL canning bottle, they were mixed with the moistened wood chips (humidity 60%) at a weight ratio of grain: wood chips = 1:10, autoclaved at 121 °C and 103 kPa for 1 h, and reautoclaved at the same temperature and pressure for 1 h after 24 h. The Trichoderma strains were inoculated into the cooled medium and cultured in the dark at 25 °C until the mycelium and spores covered the medium (Wu et al., 2019). The spores were washed with sterile water, filtered with four layers of sterile gauze, and then diluted to a concentration of 1 × 10⁸ CFU/mL with sterile water. The spore suspensions of the four Trichoderma species were mixed at equal volume to make CTA.

Effects of CTA on C. chinensis growth

The 2-year-old C. chinensis plants were rinsed with running water. Soil was sterilized at 120 °C for 3 h in oven. Plastic free-leaching pots, with a diameter of 10 cm and a total height of 10 cm, were soaked in 1‰ potassium permanganate for 1 h of disinfection, rinsed with running water, and filled with sterilized soil, 0.45 kg/pot. Then, the C. chinensis plants were transferred into the pots, with four plants/pot and 20 pots/treatment, using a completely randomized design. The following treatments were applied after 20 days when the C. chinensis plants had already adapted to the environment.

• CTA: CTA was applied to the root of C. chinensis, 15 mL/pot.

• Fer: compound fertilizer was applied to the root of C. chinensis, 2 g/L,15 mL/pot.

• H₂O: sterile water was applied to the root of C. chinensis, 15 mL/pot.

The experiments were carried out in a greenhouse under the following conditions: light 1500 Lx for 10 h/day at 20 °C, and dark 14 h/day at 15 °C (Huang & Yang, 1994). The soil was regularly watered to keep the soil moist without waterlogging. Samples were collected after 60 days. The leaves of three plants in each pot were mixed into one sample and 20 samples/treatment were prepared to determine soluble sugar, soluble protein, and chlorophyll. The remaining plants were collected, weighed, and dried at 100 °C in oven to a constant weight. The dry plants were then weighed again.

Effects of CTA on nutrients, enzyme activity, and microbial community of rhizosphere soil

Plastic free-leaching pots, with a diameter of 20 cm and a total height of 25 cm, was hung 5 cm from the ground to avoid cross affect. They were then soaked in 1‰ potassium permanganate for 1 h of disinfection, rinsed with running water, and filled with un-sterilized soil, 3.5 kg/pot. The 4-year-old C. chinensis plants with rhizosphere soil were transferred into the pots, with one plant/pot, three pots/replicate, and five replicates/treatment, using a random block design. The following treatments were applied after 20 days when C. chinensis plants had already adapted to the environment.

• CTA: CTA was applied to the root of C. chinensis, 25 mL/pot.

• Fer: compound fertilizer was applied to the root of C. chinensis, 5 g/L, 25 mL/pot.

• H₂O: sterile water was applied to the root of C. chinensis, 25 mL/pot.

The experiments were carried out in the same greenhouse as the test above. The soil was regularly watered to keep the soil moist without waterlogging. Soil samples were collected after 60 days. The roots of the plants were taken out of the soil. The soil loosely bound to the roots was shaken off, and the soil tightly bound to the roots was collected (Liu et al., 2014). The rhizosphere soil of the three plants in each replica was mixed into one sample, and 15 such soil samples were prepared to determine the nutrients, enzyme activities, and microbial community.

Determination methods

Data processing

The data were recorded in Excel 2019 and analyzed using the LSD method of one-way ANOVA and Pearson Correlation in SPSS 20.0. We considered 0.01 < P-value < 0.05 and P-value < 0.01 to be indicative of a statistically significant result.

Effects of CTA on C. chinensis growth

The soluble protein of the CTA and Fer treatments were significantly higher than the H₂O treatment (P < 0.01). The chlorophyll a, chlorophyll b, and total chlorophyll were significantly higher after the CTA treatment than after the Fer and H₂O treatments (P < 0.01) while chlorophyll a and total chlorophyll of the Fer treatment were significantly lower than the H₂O treatment (0.01 < P < 0.05). Both the fresh and dry weight were significantly higher after the CTA treatment than after the H₂O treatment (0.01 < P < 0.05) (Table 1).

Effects of CTA on rhizosphere soil nutrients and enzymatic activities

Compared with H₂O, CTA showed no significant effects on OM and available nutrients of C. chinensis rhizosphere soil (P > 0.05), while it increased the soil pH and activities of urease and catalase significantly (0.01 < P < 0.05 or P < 0.01) (Tables 2 and 3). The pH and activities of urease, catalase, and alkaline phosphatase were significantly lower after Fer than after CTA and H₂O (0.01 < P < 0.05 or P < 0.01) (Tables 2 and 3).

Effects of CTA on rhizosphere microbial communities

We obtained 1,506,050 raw tags using high-throughput sequencing of the 15 soil samples’ fungi, and 972,921 effective tags were obtained after processing, with an average efficiency of 64.60%. After clustering,19,697 OTUs were obtained. The annotated fungal OTUs of the 15 samples involved 18 phyla, 73 classes, 183 orders, 429 families, 941 genera, and 1,452 species.

We obtained1,433,061 raw tags using high-throughput sequencing of the 15 soil samples’ bacteria, and 955,153 effective tags were obtained after processing, with an average efficiency of 66.65%. After clustering, 41,597 OTUs were obtained. The annotated bacterial OTUs of the 15 samples involved 59 phyla, 147 classes, 312 orders, 417 families, 624 genera, and 300 species.

Although new OTUs still appeared when the sequencing length was more than 10,000 reads, the curve was flattened, which indicated that the sampling was reasonable and the current sequencing depth was sufficient to tell the diversity of the fungal (Fig. 1) and bacterial (Fig. 2) community contained in the sample.

Effects of CTA on rhizosphere microbial diversity

In terms of alpha-diversity, the observed species, Shannon index, Simpson index, Chao1 index, and ACE index of fungi as well as the Shannon index and Simpson index of bacteria were significantly lower after CTA than after Fer and H₂O (0.01 < P < 0.05 or P < 0.01), The bacteria/fungi (B/F) value was significantly higher after CTA than after Fer and H₂O (0.01 < P < 0.05), while no significant difference was observed between the Fer and H₂O (P > 0.05) (Table 4). With respect to the beta-diversity, the PCoA plot showed that the rhizosphere fungi and bacteria of all samples were basically divided into three groups. The PCoA plot explained 56.09% (PC1) and 11.78% (PC2) of the variation in fungal communities (Fig. 3), and 46.97% (PC1) and 18.84% (PC2) of the variation in bacterial communities (Fig. 4). The Fer and H₂O were very close to each other on PC1 and PC2, indicating similar fungal and bacterial community structures. They were both far from CTA, indicating that both the fungal and bacterial community structures of CTA were quite different from the other two treatments (Figs. 3 and 4).

Table 4:

Effects of CTA on alpha-diversity of rhizosphere microorganism.

	Treatment	CTA	Fer	H2O
Bacteria	Observed species	2,474.00 ± 111.62Aa	2,465.00 ± 59.38Aa	2,599.20 ± 127.02Aa
	Shannon index	8.62 ± 0.11Bb	8.83 ± 0.18ABa	8.89 ± 0.07Aa
	Simpson index	0.99 ± 0.00Ab	0.99 ± 0.00Aab	0.99 ± 0.00Aa
	Chao1 index	2,707.95 ± 130.84Aa	2,706.73 ± 49.43Aa	3,220.77 ± 978.90Aa
	ACE index	2,747.85 ± 133.42Aa	2,742.66 ± 45.26Aa	3,005.80 ± 400.64Aa
Fungi	Observed species	977.60 ± 166.35Ab	1,248.80 ± 188.88Aa	1259.00 ± 109.31Aa
	Shannon index	4.56 ± 0.48Bb	6.54 ± 0.55Aa	6.57 ± 0.12Aa
	Simpson index	0.81 ± 0.05Bb	0.96 ± 0.01Aa	0.97 ± 0.00Aa
	Chao1 index	1,116.93 ± 206.27Ab	1,388.65 ± 203.62Aa	1,393.60 ± 147.92Aa
	ACE index	1,155.18 ± 214.03Ab	1,407.73 ± 201.51Aab	1,433.51 ± 152.72Aa
	B/F	2.60 ± 0.49Aa	2.01 ± 0.33Ab	2.08 ± 0.23Ab

DOI: 10.7717/peerj.15652/table-4

Note:

Data are reported as Mean ± SD. Different letters indicate significant differences among different treatments. Capital letters: P < 0.01; lower-case letters: 0.01 < P < 0.05. CTA, compound Trichoderma agent; Fer, compound fertilizer; H₂O, sterile water.

The fungal Venn plot showed that the three treatments had 1,298 common OTUs. There were many more unique OTUs of Fer and H₂O than CTA (about two times as much), indicating that the fungal diversities of Fer and H₂O were higher than that of CTA. Fer and H₂O shared the most OTUs, and both of them shared few OTUs with CTA, indicating that the fungal community structures of Fer and H₂O were similar to each other, but different from that of CTA. This was consistent with the aforementioned diversity analysis results (Fig. 5). The bacterial Venn plot showed that the three treatments had 2,735 common OTUs. There were fewer unique OTUs of Fer and H₂O than CTA. Fer and H₂O shared more OTUs, and both of them shared fewer OTUs with CTA (Fig. 6).

Effects of CTA on the rhizosphere microbial community structure

The rhizosphere fungi of the three treatments were different at the phylum, genus, and species levels. Ascomycota, Mortierellomycota, Basidiomycota, etc. were the dominant phyla of rhizosphere fungi (Fig. 7). The relative abundance of Ascomycota was significantly higher (P < 0.01) while Basidiomycota was significantly lower after CTA than after H₂O and Fer (P < 0.01). There were also significant differences among the relative abundance of Chytridiomycota, Glomeromycota, Rozellomycota, etc. in the three treatments (0.01 < P < 0.05 or P < 0.01) (Fig. 8).

The dominant fungal genera included Trichoderma sp., Fusarium sp., Sporothrix sp., Trichocladium sp., etc. (Fig. 9). The relative abundance of Trichoderma sp. was significantly higher after CTA than after H₂O and Fer (P < 0.01). There were also significant differences among the relative abundance of Solicoccozyma sp., Octaviania sp., Clavulina sp., etc. in the three treatments (0.01 < P < 0.05 or P < 0.01) (Fig. 10).

The dominant fungal species were T. hamatum, Sporothrix nigrograna, Fusicolla merismoides, Trichocladium griseum, etc. (Fig. 11). The relative abundance of T. hamatum and Trichoderma sp. was significantly higher after CTA than after H₂O and Fer (P < 0.01). The relative abundance of Ilyonectria mors-panacis was significantly lower after CTA than after Fer (0.01 < P < 0.05). There were also significant differences among the relative abundance of Octaviania hesperi, Solicoccozyma terrea, etc. in the three treatments (0.01 < P < 0.05 or P < 0.01) (Fig. 12).

The dominant bacterial phyla included Proteobacteria, Actinobacteriota, Firmicutes, etc. (Fig. 13). There were significant differences among the relative abundance of Actinobacteriota, Chloroflexi, Gemmatimonadetes, etc. in the three treatments (0.01 < P < 0.05 or P < 0.01) (Fig. 14).

The dominant bacterial genera were Bryobacter sp., Candidatus Solibacter sp., Bradyrhizobium sp., etc. (Fig. 15). The relative abundance of Corynebacterium sp. was significantly lower after CTA than after H₂O (0.01 < P < 0.05). There were also significant differences among the relative abundance of Ralstonia sp., Bryobacter sp., Rhodanobacter sp., etc. in the three treatments (0.01 < P < 0.05 or P < 0.01) (Fig. 16).

The dominant bacterial species were Ralstonia pickettii, Bradyrhizobium elkanii, Pasteurellaceae bacterium, etc. (Fig. 17). The relative abundance of R. picketti was significantly higher after CTA than after H₂O and Fer (P < 0.01). There were also significant differences among the relative abundance of Pasteurellaceae bacterium, delta proteobacterium WX81, bacterium Ellin6089, etc.in the three treatments (0.01 < P < 0.05 or P < 0.01) (Fig. 18).

Correlation analysis

Correlations were found among the C. chinensis rhizosphere soil nutrients, enzymatic activities, and the top ten fungal and bacterial species in relative abundance. Soil alkali-hydrolysable N and available K contents were significantly negatively correlated with pH and urease, alkaline phosphatase, and catalase activities (P < 0.01) and were significantly positively correlated with relative abundance of B. elkanii, delta proteobacterium WX81, etc. (0.01 < P < 0.05 or P < 0.01). The activities of urease, alkaline phosphatase, and catalase were significantly positively correlated with each other (P < 0.01). The pH was significantly positively correlated with the activities of urease, alkaline phosphatase, and catalase (P < 0.01). The activities of urease, catalase, alkaline phosphatase, and pH were significantly positively correlated with the relative abundance of two Trichoderma fungi (0.01 < P < 0.05 or P < 0.01). The relative abundance of two Trichoderma fungi was significantly positively correlated with that of R. picketti (P < 0.01), and significantly negatively correlated with those of delta proteobacteriumWX81 and Mortierella sp. (0.01 < P < 0.05) (Table 5).

Effects of CTA on C. chinensis growth

Many studies have shown that Trichoderma fungi can promote plant growth. When T. asperellum and T. harzianum were applied to cucumber seedlings, the plant heights, stem diameters, leaf areas, whole plant fresh weights, and chlorophyll contents were all significantly higher than those of the control (Guangshu et al., 2021). Trichoderma fungi work by releasing auxin (López-Bucio, Pelagio-Flores & Herrera-Estrella, 2015), activating soil nutrition and enzymes (Fu et al., 2021). Soluble protein, chlorophyll, and weight are the indices of plant growth. Compared with H₂O, CTA can increase individual weight, soluble protein, and chlorophyll content while compound fertilizer decreased chlorophyll content in leaf. CTA performed better than compound fertilizer.

Effects of CTA on rhizosphere soil nutrients and enzyme activities

Many studies have reported on the soil improvements caused by Trichoderma fungi: increased enzyme activities of soil, adsorption of heavy metals, and adjustment of soil acidity (Fu et al., 2021). Soil enzymes are essential indicators of soil fertility and are involved in soil material and energy metabolism. The sources of soil enzymes mainly include plant root exudates, soil microbial exudates, animal releases, and the decomposition of animal and plant residues (Jiang et al., 2009). Urease hydrolyzes urea and promotes the transformation of N fertilizer, showing the N supply capacity of the soil (Li et al., 2018b). Catalase decomposes H₂O₂, which is produced during plant nutrient metabolism, into H₂O and O₂, thereby alleviating the toxicity of hydrogen peroxide to soil organisms (Chu, Zhang & Shi, 2015; Visser & Parkinson, 1992). Phosphatase hydrolyzes soil organic P and improves P availability (Xu et al., 2020). Alkaline phosphatase activity is also one of the main soil factors affecting the growth of C. chinensis (Duan et al., 2020). Soil pH affects the activities of soil enzymes, nutrient availability, plant growth, and microbial community structure, and is a crucial indicator of soil fertility (Jiang, 2018). Soil acidification lowers nutrient availability, inhibits soil enzyme activity, reduces microbial diversity, damages plant roots, and eventually compromises crop yield and quality (Liang, 2021).

The results of this study revealed that artificially added CTA could increase the activities of urease and catalase in the C. chinensis rhizosphere soil, while compound fertilizer decreased the activities of urease, catalase, and alkaline phosphatase, which was consistent with the previous study. After T. hamatum was applied to mung bean, the activities of soil urease, alkaline phosphatase, catalase, and sucrase were all elevated (Baazeem et al., 2021). The activities of sucrase, acid phosphatase, urease, and catalase in the rhizosphere soil of Pinus sylvestris seedlings applied with Trichoderma harzianum and Trichoderma virens were all significantly higher than those in the control (Halifu et al., 2019), while the activities of sucrase, urease, and alkaline phosphatase in the rhizosphere soil of pepper applied with synthetic fertilizer alone were lower than those in the soil treated with Trichoderma organic fertilizer, partially replacing synthetic fertilizer (Jin et al., 2021).Therefore, Trichoderma increased the enzyme activities of the soil, while synthetic fertilizer inhibited them. Trichoderma spp. enlarged the contact area between the root and the soil and boosted the secretion of extracellular enzymes such as sucrase and urease, so as to improve the activities of soil enzymes (Halifu et al., 2019). In addition, Trichoderma elevated the enzyme activities by increasing soil pH, while the compound fertilizer decreased pH, possibly due to the induced soil acidification that inhibited the enzyme activities (Liang, 2021).

CTA significantly increased soil pH. The application of T. harzianum to Brassica campestris altered the soil pH from 6.75 to 6.97 (Liu et al., 2020), while Trichoderma reduced pH in saline-alkali soil (Jian et al., 2021). This indicated that Trichoderma had a two-way regulating effect on soil pH. Presumably, application of a large amount of Trichoderma to the soil affected the balance of the soil microbial communities, which affected the root exudates and thus changed the soil pH (Wang, 2014). However, compound fertilizer increased soil acidity. Numerous other studies have also shown that the long-term application of synthetic fertilizers, especially N fertilizers, led to soil acidification (Tian & Niu, 2015). The soil pH in most C. chinensis-producing areas was acidic (Chen et al., 2005), and the ideal soil pH for C. chinensis was 5.5–7.0 (Sun, 2006). Therefore, compared with compound fertilizer, CTA performed better at adjusting the soil pH to the ideal range for C. chinensis.

CTA could not improve soil nutrient and OM content. However, the combination of CTA and organic fertilizers could overcome this drawback. When T. longibrachiatum was applied to mangoes in combination with organic fertilizer, the yield increased by 13% compared to using no fertilizer, 7% compared with synthetic fertilizer applied alone, and 6% compared with organic fertilizer applied alone (Zhu et al., 2021).

Effects of CTA on soil microbial community structure and function

Microorganisms are an important part of the soil and play a critical role in the decomposition of OM and nutrient cycling (Jiao et al., 2019). They also affect the physicochemical properties of the soil (Sharma et al., 2017). The diversity of soil microorganisms affects soil fertility (Tahat et al., 2020).

In this study, both the number and alpha-diversity of rhizosphere fungi were lower after CTA than after the other two treatments. It was also found that the alpha-diversity and number of rhizosphere fungi of P. sylvestris seedlings treated with T. harzianum and T. virens were lower than those of the control (Halifu et al., 2019). The alpha-diversity of bacteria was lower after CTA than after the other two treatments. Yu et al. (2020) found that, after Trichoderma brevicompactum treatment, the alpha-diversity and number of rhizosphere bacteria were lower than those of the control. This demonstrated the inhibitory effect of Trichoderma on other microorganisms, which is related to its biocontrol function. Trichoderma spp. grow fast, can occupy the growing space, and absorb the required nutrients quickly. At the same time, Trichoderma spp. may produce cell wall-degrading enzymes to break down microbial cells in the soil environment to absorb nutrients (Zhang, 2015).

CTA increased rhizosphere soil B/F value. It was also found that Trichoderma bio-organic fertilizer increased rhizosphere soil B/F value (Pu, 2019). Soil B/F value is usually closely related to soil-borne diseases and continuous cropping obstacles. Continuous cropping tends to increase fungi and decrease bacteria in soil, which causes soil-borne diseases. For example, after continuous cropping of cucumber, the soil turns from a bacterial type to fungal type (Du et al., 2017). It was concluded that bacterial-type soil was an indicator of higher fertility and vitality, while the fungal-type soil was a sign of soil failure (Feng et al., 1999). Therefore, CTA improved the soil microbial community.

CTA significantly altered the microbial community structure in the soil at the phylum, genus, and species levels. In this study, the relative abundance of Ascomycota was significantly higher, and the relative abundance of Basidiomycota was significantly lower after CTA than after H₂O and Fer. It was revealed that healthy soil contained a higher abundance of Ascomycota, while sub-healthy soil had more abundant Basidiomycota (Qiao, 2021), indicating that CTA-treated soil was healthier than H₂O-or Fer-treated soil. CTA decreased the abundance of harmful Corynebacterium sp. (Guo et al., 2002), and I. mors-panacis (Farh, Kim & Singh, 2017) in the soil. Ilyonectria sp. is comprised of various phytopathogenic fungi (Bischoff Nunes & Goodwin, 2022), including the pathogen responsible for the root rot disease of C. chinensis (Wu et al., 2021). A large number of studies showed that Trichoderma could reduce the number of various phytopathogenic microorganisms in the soil, thereby controlling the disease. When Trichoderma Gamsii and T. harzianum were tested against Fusarium Pseudograminearum, a significant decrease in crown rot symptoms was achieved in wheat, with reduced pathogen populations in soil (Stummer et al., 2022). CTA increased the abundance of Trichoderma spp. and R. picketti which could degrade phenolic acid, an allelopathic compound in the soil, that can control continuous cropping obstacles of C. chinensis.

The Tichoderma spp. were still dominant in C. chinensis rhizosphere soil 60 days after application of CTA, showing a long lifetime. Other studies also revealed that Tichoderma can exist in soil for a long time. The total valid term of rhizosphere Tichoderma atroviride applied to loquat was 75 days (Lu et al., 2020). When Trichoderma asperellum was applied to cucumber, its content in rhizosphere soil reached its peak at 70 d (Huo et al., 2016).

There were differences among the microbial functions of the three treatments. CTA decreased the abundance of Pathotroph, which showed that CTA inhibited some pathogenic fungi, and increased the abundance of Saprotroph, due to the fact that Trichoderma are Saprophytic fungi.

Correlation among rhizosphere soil nutrients, enzyme activities, and microbial community structure

An significant positive correlation was found between pH and urease, alkaline phosphatase, and catalase activities. Acosta-Martínez & Tabatabai (2000) also found that the activities of urease, amidase, alkaline phosphatase, and phosphodiesterase were significantly positively correlated with soil pH, which indicated that the increase in soil pH within a certain range benefited the soil enzymes, while soil acidification inhibited enzyme activity (Zhao et al., 2022). This was due to the pH which directly affected the speed of soil enzymes participating in biochemical reactions. When the pH was beyond the ideal range, it inhibited enzyme activities (Zhao, 2011). At the same time, the activities of urease, catalase, and alkaline phosphatase, as well as pH, were all significantly positively correlated with the abundance of two Trichoderma fungi, indicating that the application of CTA elevated pH and enzyme activities.