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Article

Turning Waste into Wealth: Utilizing Trichoderma’s Solid-State Fermentation to Recycle Tea Residue for Tea Cutting Production

by
Zhen Meng
1,
Shuangshuang Xiang
1,
Xue Wang
1,
Jian Zhang
1,2,
Guoxin Bai
3,
Hongjun Liu
1,4,*,
Rong Li
1 and
Qirong Shen
1
1
Jiangsu Provincial Key Laboratory for Solid Organic Waste Utilization, Key Laboratory of Organic-Based Fertilizers of China, Jiangsu Collaborative Innovation Center for Solid Organic Wastes, Educational Ministry Engineering Center of Resource-Saving Fertilizers, Nanjing Agricultural University, Nanjing 210095, China
2
Key Laboratory of Green Intelligent Fertilizer Innovation, MARD, Sinong Bio-Organic Fertilizer Institute, Nanjing 210000, China
3
Comprehensive Service Center of Guanlin Town, Wuxi 214251, China
4
College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(3), 526; https://doi.org/10.3390/agronomy14030526
Submission received: 1 February 2024 / Revised: 29 February 2024 / Accepted: 1 March 2024 / Published: 4 March 2024
(This article belongs to the Section Agricultural Biosystem and Biological Engineering)
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Abstract

:
Trichoderma is a widely recognized plant-growth-promoting fungus that has been extensively utilized in various agricultural applications. However, research on the economic production of Trichoderma spores and their effects on tea cuttings must be further advanced. In this study, T. guizhouense NJAU 4742 (NJAU 4742) emerged as a growth-promoting strain for tea cuttings, and the spore-production conditions of NJAU 4742 attained through solid-state fermentation (SSF) using tea residues were optimized. In a pot experiment, nursery substrates containing different concentrations of NJAU 4742 spores were tested for their influence on tea cutting growth and the rhizosphere fungal community. The optimal conditions for spore yield were determined as a 7:3 (w/w) ratio of tea residue to rice bran, a material thickness of 3 cm, an inoculum concentration of 15% (v/w), and an incubation time of 4 days, resulting in a spore count of 1.8 × 109 CFU/g. Applying NJAU 4742 spore products significantly increased the biomass of tea cuttings and influenced the fungal community composition. Moreover, higher concentrations of NJAU 4742 spores yielded better growth performance, and applying nursery substrate with 1.0 × 107 CFU/mL spores was the most economically viable option. Notably, among the top ten fungal genera with the highest relative abundance, Trichoderma showed a positive correlation with the fresh weight of tea cuttings, while the others exhibited a negative correlation. Overall, utilizing tea residue for SSF to produce NJAU 4742 was a feasible approach, and the application of NJAU 4742 spores enhanced the growth of tea cuttings by increasing the relative abundance of Trichoderma.

1. Introduction

The excessive use of fertilizers and pesticides can cause significant harm to the environment. However, plant-growth-promoting fungi (PGPF) offer a sustainable and environmentally friendly alternative [1,2,3]. Trichoderma, widely present in various environments, is one of the most commonly utilized PGPFs [4,5]. Trichoderma can directly or indirectly suppress various harmful organisms to promote plant growth through mycoparasitic interactions [6], the production of secondary metabolites (such as hydrolytic enzymes and peptaibol antibiotics) that inhibit target organisms [7], ecological niche and nutritional competition [8], as well as the induction of plant resistance [9]. Additionally, Trichoderma can promote plant growth by producing plant growth-regulating substances (such as auxins, ethylene, and other volatile organic compounds) and enhancing nutrient cycling [10,11]. However, the effectiveness of Trichoderma in promoting plant growth relies on the presence of a high number of spores in the soil [12,13]. Therefore, achieving a high concentration of Trichoderma spores is crucial.
Solid-state fermentation (SSF) is a biotechnological process involving solid substrates with minimal or no free water. SSF is commonly employed for the production of various value-added products from filamentous fungi, including enzymes, antibiotics, proteins, conidia, biofuels, and animal feed [14,15,16]. It provides a cost-effective and convenient method for mass-producing fungal spores compared to submerged fermentation [17]. Moreover, substrates commonly used for SSF are agricultural and industrial residues, such as crop residues, fruit and vegetable peels, and spent grains, which facilitate resource utilization and environmental conservation [18]. The choice of substrate substantially influences the outcome of fermentation [19]. Of various agro-industrial residues, rice husk, potato peel, beer draff, and orange pomace resulted in the highest spore production of Trichoderma harzianum [20]. Therefore, selecting appropriate substrates is crucial for Trichoderma sporulation.
Tea (Camellia sinensis L.), originating from China, is one of the most popular nonalcoholic beverages worldwide and is enjoyed by approximately 3 billion people [21,22]. In 2020, global tea consumption reached approximately 6.3 million tons and is projected to increase to 7.4 million tons by 2025 [23]. Similarly, the retail value of tea is predicted to reach USD 73 billion by 2024 [24]. China, as the largest tea-producing country, accounts for approximately 40% of the total global tea production [25]. During tea production and consumption, approximately 90% of tea becomes tea residue [26]. Tea residue is generated from restaurants, hotels, teahouses, and other places, causes environmental pollution, and demands extensive labor for its disposal [27]. Tea residue is a valuable waste resource comprising cellulose, hemicellulose, lignin, polyphenols, proteins, and tannins [28,29]. Currently, tea residue is utilized for various applications, including the extraction of polyphenols and polysaccharides, biochar production, silage, composting, and fermentation [30,31,32,33]. Among these applications, using tea residue for composting or fermentation is a cost-effective approach [34].
Trichoderma possesses strong lignocellulose-degrading capabilities and act as aggressive bio-degraders even under nutrient-limiting conditions [35,36]. Trichoderma are typically employed in composting and SSF to enhance degradation efficiency and produce value-added products [37,38,39]. He et al. [40] inoculated composite microbes containing Trichoderma during biogas residue composting, resulting in increased degradation rates of cellulose, hemicellulose, and lignin compared to the control group. Hamrouni et al. [41] optimized the SSF conditions for Trichoderma asperellum to produce 6-pentyl-alpha-pyrone. The products containing Trichoderma had been proven to enhance cucumber biomass; improve the growth, yield, and quality of Bupleurum chinense; and enhance nutrient absorption in Phaseolus vulgaris while reducing disease incidences caused by Sclerotium rolfsii [42,43,44]. Despite extensive research conducted on the beneficial effects of Trichoderma on various plants, studies on its fermentation of tea residue and its potential for promoting tea cutting growth are limited.
The plant rhizosphere microbiome, often referred to as the plant’s second genome, is closely associated with plant health [45]. Rhizosphere microorganisms play crucial roles in nutrient transformation, organic matter decomposition, pathogen suppression, and the promotion of plant growth [46]. The application of bio-ameliorants may lead to changes in the structure and function of rhizosphere microorganisms, thereby resulting in alterations in plant traits [47,48]. High-throughput sequencing is a milestone technology in the development of DNA sequencing and microbial community research [49]. This method has the capability to greatly reveal the specific composition characteristics of complex environmental microbial communities and has been widely applied in studying the effects of bio-ameliorants on soil and rhizosphere microbial communities [50]. Utilizing high-throughput sequencing could help us clarify the impact of applying Trichoderma on the rhizosphere microbiome of tea cuttings.
In this study, we compared the growth-promoting abilities of three Trichoderma strains (Trichoderma guizhouense NJAU 4742, T. longiformis MD30, and T. orange JS84) on tea cuttings. Moreover, the sporulation conditions in the SSF of the best growth-promoting strain were optimized using tea residue as the substrate. Additionally, we evaluated the effects of different concentrations of NJAU 4742 spores on the growth of tea cuttings and identified dynamic changes in the fungal community amended by NJAU 4742 spores through high-throughput sequencing. The main objectives of this research were as follows: (1) to produce NJAU 4742 spores through SSF using tea residue; (2) to assess the effects of NJAU 4742 spores on the growth of tea cuttings; and (3) to identify dynamic changes in the fungal community amended by NJAU 4742 spores using high-throughput sequencing.

2. Materials and Methods

2.1. Microorganism and Culture Conditions

Trichoderma guizhouense NJAU 4742 (China General Microbiology Culture Collection Center Accession No. 3308) was isolated from a mature compost sample, and its genome sequence has already been published in the NCBI database (accession number LVVK00000000.1). Trichoderma longiformis MD30 (China General Microbiology Culture Collection Center Accession No. 17467) and Trichoderma orange JS84 (China General Microbiology Culture Collection Center Accession No. 17466) were isolated from agricultural soils collected from tropical areas [51,52]. The three Trichoderma strains were obtained from the Jiangsu Provincial Key Laboratory of Organic Solid Waste Utilization, Nanjing, China. The mycelium and spore suspensions of the three Trichoderma species were prepared following the method described by Liu et al. [53]. In summary, three preserved Trichoderma strains were inoculated onto potato dextrose agar (PDA, Oxoid, UK) at 28 °C for 7 days for activation. The concentrations of activated Trichoderma spores were adjusted to 108 CFU/mL using sterile water and a hemocytometer. Subsequently, spore suspensions were inoculated into PDA liquid medium (1%, v/v) and cultured at 28 °C and 170 r/min for 4 days to obtain Trichoderma suspensions containing mycelia and spores.

2.2. Growth-Promoting Test of Different Trichoderma Strains

The growth-promoting test was conducted in a greenhouse with an average temperature of 25 °C and a humidity of 80% (118°85′ E, 32°04′ N, and an elevation of 9 m) at Nanjing Agricultural University from October 2020 to December 2020 to identify Trichoderma strains that could promote tea cutting growth for the optimization of SSF conditions. The experiment was carried out in seedling trays using nursery substrates. It included four treatments: CK (no Trichoderma spore), NJAU 4742 (applied nursery substrate with 5.0 × 107 CFU/mL FW NJAU 4742 spores), JS84 (applied nursery substrate with 5.0 × 107 CFU/mL FW JS84 spores), and MD30 (applied nursery substrate with 5.0 × 107 CFU/mL FW MD30 spores). The Trichoderma spores obtained from PDA liquid medium fermentation were diluted with sterile water to achieve the target concentration and added to nursery substrates. Each treatment contained thirty tea cuttings (Camellia sinensis cv. Suchazao). The composition of the nursery substrate was prepared using turf, red soil, vermiculite, and perlite at a volumetric ratio of 3:3:2:2. On the 45th day, the lengths of the tea cuttings’ buds were measured, and the survival rate was calculated on the 90th day.

2.3. Optimization of SSF Parameters for NJAU 4742

Several single-factor optimization studies were conducted to maximize the spore production of NJAU 4742 through SSF using tea residue. The optimization process involved determining the material ratio of tea residue to rice bran (60, 70, 80, 90, and 100% w/w), material thickness (2, 3, 4, 5, and 6 cm), inoculum concentration of NJAU 4742 suspension (5, 10, 15, 20, and 25% v/w), and incubation time (2, 3, 4, 5, and 6 days). The material was sterilized at 115 °C for 30 min, then cooled to approximately 25 °C and inoculated with NJAU 4742 suspension. Each treatment consisted of three plates, with 1000 g of fresh material in each plate, and SSF was conducted at 28–30 °C. After fermentation, a 5 g sample of the fermented product was transferred into a 50 mL centrifuge tube, diluted to a suitable concentration with sterile water, homogenized with a vortex (Vortex-Genie® 2; Scientific Industries, Bohemia, New York, USA) mixer for even distribution, and the spore concentration was quantified utilizing a hemocytometer.

2.4. Pot Experiment Design and Nursery Substrate Sampling

The greenhouse pot experiment was conducted in a greenhouse with an average temperature of 25 °C and a humidity of 80% (118°85′ E, 32°04′ N, and an elevation of 9 m) at Nanjing Agricultural University from December 2020 to May 2021 to investigate the effects of different concentrations of NJAU 4742 spores fermented by tea residue on the growth of tea cuttings. The experiment included five treatments: CK (no NJAU 4742 spores), T1 (applied nursery substrate with 1.0 × 108 CFU/mL FW NJAU 4742 spores), T2 (applied nursery substrate with 5.0 × 107 CFU/mL FW NJAU 4742 spores), T3 (applied nursery substrate with 1.0 × 107 CFU/mL FW NJAU 4742 spores), and T4 (applied nursery substrate with 5.0 × 106 CFU/mL FW NJAU 4742 spores). The SSF fermentation products of NJAU 4742 were diluted with sterile water to obtain the target concentration of spore suspensions and added to nursery substrates. Each treatment had five replicates. The plant height and stem diameter of the tea cuttings were regularly measured, and the fresh weight and root length were measured on the 45th day. Nursery substrate samples were collected after harvest following the method described by Liu et al. [54].

2.5. DNA Extraction and Illumina MiSeq Sequencing

The total DNA of each nursery substrate sample was extracted using Power Soil DNA Isolation Kits (MoBio Laboratories, Carlsbad, CA, USA) following the manufacturer’s instructions. The quality and concentration of the extracted DNA samples were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Subsequently, all DNA samples were utilized as templates for amplifying the ITS region using the primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′), as previously described by Li et al. [55]. The amplification and sequencing of the ITS genes were performed by Personal Biotechnology Co., Ltd. (Shanghai, China) using an Illumina MiSeq instrument (San Diego, CA, USA).

2.6. Bioinformatic Analysis

The sequencing data obtained from each sample were split according to the unique barcodes and trimmed of the adaptor and primer sequences using QIIME 2 2021.8 software [56]. After the removal of low-quality sequences, the forward and reverse sequences for each sample were merged. Following the standard operating procedures of UPARSE [57], the data were processed using USEARCH and Perl scripts to generate operational taxonomic unit (OTU) tables for the sequencing samples. We ultimately obtained 1326 fungal OTUs, totaling 3,001,080 sequences, with a minimum of 46,300 and a maximum of 208,197. Finally, representative sequences of fungal OTUs were selected [57] and classified using the RDP classifier against the UNITE Fungal ITS database [58]. Alpha diversity, including the Shannon index, Chao1 estimator, and Pielou index, was analyzed and compared in the R environment (version 3.6.6) using the “vegan” package. To assess the variations in fungal community structures, principal coordinate analysis (PCoA) based on the Bray–Curtis distance was employed. Furthermore, permutational analysis of variance (PERMANOVA) was conducted to evaluate the statistical significance of differences in community structures. Additionally, Pearson’s correlation coefficient was employed to evaluate the correlation between the fresh weight of tea cuttings and the relative abundance of the top ten fungal genera.

2.7. Statistical Analysis

Statistical analysis was performed using Microsoft Excel 2021 (Microsoft Inc., Redmond, WA, USA) and SPSS 23.0 (SPSS Inc., Chicago, IL, USA). A one-way ANOVA was employed to analyze the variance among the different treatments. To assess the significance of differences between treatments, Duncan’s method was utilized, with a significance level set at p < 0.05.

3. Results

3.1. Effects of Different Trichoderma Strains on the Growth of Tea Cuttings

In comparison to MD30 and JS84, NJAU 4742 demonstrated significant improvements (p < 0.05) in both the survival rate (Figure 1A) and bud length (Figure 1B) of tea cuttings. On the other hand, neither MD30 nor JS84 showed significant improvements (p > 0.05) in the biomass of tea cuttings compared to the control group (CK). These results indicated that NJAU 4742 has the ability to promote the growth of tea cuttings, as evidenced by its positive effects on both the survival rate and bud length. In contrast, MD30 and JS84 did not exhibit significant growth-promoting effects on tea plant biomass.

3.2. Optimized Parameters of SSF for NJAU 4742

Figure 2 presents the effects of different parameters on the spore production of NJAU 4742 in SSF. The results indicated that a material ratio of 70% tea residue and 30% rice bran resulted in the highest spore production compared to other treatments (Figure 2A). The optimal material thickness was determined to be 3 cm (Figure 2B). Although there was no significant difference in spore numbers between a 15% and 20% inoculum concentration, spore production was higher at the 15% inoculum concentration than other treatments (Figure 2C). In terms of incubation time, the spore counts significantly increased with time until day 4 (Figure 2D). In summary, the optimal parameters for the SSF process of NJAU 4742 were as follows: a material ratio of seven parts tea residue to three parts rice bran, a material thickness of 3 cm, a 15% inoculum concentration, and an incubation time of 4 days. These parameters resulted in a maximum of 1.8 × 109 CFU/g FW for producing NJAU 4742 spores.

3.3. Effects of Different Concentrations of NJAU 4742 Spores on the Growth of Tea Cuttings

The effects of different concentrations of NJAU 4742 spores on the growth of tea cuttings are shown in Figure 3. Compared to the CK (with no NJAU 4742 application) and T4 (5.0 × 106 CFU/mL FW NJAU 4742 application) treatments, T1 (1.0 × 108 CFU/mL FW NJAU 4742), T2 (5.0 × 107 CFU/mL FW NJAU 4742), and T3 (1.0 × 107 CFU/mL FW NJAU 4742) significantly (p < 0.05) increased the fresh weight (Figure 3A) and stem diameter (Figure 3B) of tea cuttings. Furthermore, the T1, T2, and T3 treatments also significantly (p < 0.05) increased the plant height (Figure 3C) and root length (Figure 3D) of tea cuttings compared to those of the CK treatment, while there was no significant difference between those of the T3 and T4 treatments. In summary, T1 showed the highest values of fresh weight, stem diameter, plant height, and root length. However, no significant difference was observed among T1, T2, and T3 treatments. These results suggested that applying higher concentrations of NJAU 4742 spores led to better growth promotion performance in tea cuttings. Among the treatments, 1.0 × 107 CFU/mL FW was the most economically viable option for promoting tea cutting growth.

3.4. Effects of Different Concentrations of NJAU 4742 Spores on Fungal Community Composition

The analysis of the Chao1 index did not show any significant differences among all treatments (Figure S1A). However, regarding the fungal Shannon index, the T1 treatment group reported significantly decreased values compared to the T4 treatment, while the other treatments did not have a significant effect (Figure S1B). The results of PCoA based on the Bray–Curtis distance metric revealed significant differences (R2 = 0.3998, p < 0.001) in the fungal communities among the different treatments (Figure 4). The results indicated that the treatment furthest from the CK was T4, followed by T3, T2, and T1. This suggested that the application of higher concentrations of NJAU 4742 spores resulted in greater variation in the fungal community composition.

3.5. Relationships between Fungal Community Composition and Fresh Weight of Tea Cuttings

Figure 5 shows the taxonomic community structure at the genus level of fungi under different treatments. The top ten most abundant fungal genera were Trichoderma, Boothiomyces, Mortierella, Conlarium, Phialemonium, Humicola, Geminibasidium, Thermomyces, Gymnostellatospora, and Ovatospora, which collectively accounted for approximately 90% of the fungal sequences. Among them, Trichoderma exhibited an increasing trend in relative abundance with the addition of NJAU 4742. Compared to the CK, the relative abundance of Trichoderma increased from 2.35% to 84.27% in T1, 57.25% in T2, 37.36% in T3, and 25.67% in T4. (Figure 5A). Furthermore, there was a significant positive correlation between the relative abundance of Trichoderma and the fresh weight of tea cuttings (r = 0.64, p = 0.00019, Figure 5B). Conversely, the remaining nine genera (Boothiomyces (r = −0.018, p = 0.93), Mortierella (r = −0.25, p = 0.118), Conlarium (r = −0.25, p = 0.19), Phialemonium (r = −0.55, p = 0.0018), Humicola (r = −0.33, p = 0.079), Geminibasidium (r = −0.52, p = 0.0041), Thermomyces (r = −0.54, p = 0.0027), Ovatospora (r = −0.46, p = 0.012), and Gymnostellatospora (r = −0.5, p = 0.0052)) demonstrated a negative correlation with the fresh weight of tea cuttings (Figure S2).

4. Discussion

According to Cai and Druzhinina [59], as of July 2020, there are 375 Trichoderma species with valid names. However, different Trichoderma species exhibit varying characteristics, leading to diverse effects on plants. Liu et al. [60] specifically selected three Trichoderma strains with notable growth rates and spore production for their experiment, revealing different preventive methods against Syringa powdery mildew. Similarly, Hao et al. [61] conducted a cucumber growth-promoting experiment involving 15 Trichoderma strains, highlighting the best growth-promotion effect of strain T-10264. In our study, the application of NJAU 4742 spores significantly enhanced the survival rate and bud length of tea cuttings compared to two other spores of Trichoderma strains with equivalent concentrations. These findings underscored NJAU 4742 as a promising Trichoderma strain for the growth promotion of tea cuttings, prompting the selection of NJAU 4742 for further investigation.
Through the optimization of fermentation conditions, the count of spores obtained in this study was higher than that obtained by Sun et al. [62] using biogas residue to ferment NJAU 4742. This difference may be attributed to the different materials. The presence of unused tea polyphenols, crude protein, and lignocellulose in tea residue is favorable for NJAU 4742 sporulation [63]. Similar to our study, the maximum spore production of T. harzianum in SSF using green waste as material was 3.0 × 109 CFU/g [64]. Material thickness is critical in regulating oxygen content and temperature during fermentation. Liu et al. [53] reported the maximum sporulation at a material thickness of 3 cm, similar to our study. Zhang and Yang [65] reported that a low inoculum concentration was not conducive to spore production, while a high inoculum concentration led to spore death due to toxin production. Thus, an inoculum concentration of 15% was deemed appropriate in this study. Considering the time cost and spore activity, the spore count increased slowly after the 4th day, indicating that a 4-day incubation time was suitable, which is consistent with the strategy employed by Zhang et al. [66]. In China, Trichoderma spores with a concentration of 1.0 × 109 CFU/g are typically priced at CNY 10,000 (approximately USD 1400) per ton. Our spore product, with a concentration of 1.8 × 109 CFU/g, significantly enhanced the value of tea residue. It was worth noting that this study optimized the SSF conditions of Trichoderma with spore count as the target product. Sporulation was easily obtained during SSF, but the resulting products had a limited shelf life. As the mechanisms of action of microorganisms on plants are elucidated, the production of higher value-added products such as antibiotics, enzymes, and proteins may be the future trend of SSF development [67,68].
When a certain quantity of spores was applied, Trichoderma strains exhibited opportunistic behavior as plant symbionts, stimulating plant growth [69]. It was documented that T. harzianum SQR-T037 enhanced root length and tips in tomato plants via phytohormone secretion [70]. Liu et al. [71] reported that the use of biological organic fertilizers containing NJAU 4742 improved soil enzyme activity, enhanced soil nutrient utilization by peppers, and stimulated pepper growth. Meng et al. [72] indicated that TgSWO from NJAU 4742 may contribute to promoting cucumber growth by altering root morphology and cell wall architecture. In our study, the application of NJAU 4742 spores significantly increased the tea cutting biomass, similar to previous studies [73]. As previously mentioned, various direct mechanisms may underlie this growth-promoting effect. In addition to direct effects, the addition of bio-amendments can also alter the microbial community structure in the plant rhizosphere. The rhizosphere microbiome is an important contributor to plant growth, with significant changes in microbial community structure closely related to plant growth and yield [74]. Hang et al. [75] found that utilizing bio-organic fertilizer amended with NJAU 4742 led to a significant change in the fungal community rather than the bacterial community and was correlated with a significant increase in cucumber yield. Moreover, the soil slurry experiment validated the growth-promoting ability of the fungal community. Miao et al. [76] found that long-term application of compost altered fungal community composition, enhancing fungal decomposition of cellulose-containing substrates, thereby promoting increased availability of nutrients for plants. Fu et al. [77] demonstrated that application of Trichoderma can regulate salt ion balance in saline-alkaline soils, increasing the relative abundance of beneficial fungi in fungal communities and reducing the relative abundance of pathogens, ultimately leading to improved maize yield. In our study, the higher biomass of tea cuttings after the application of NJAU 4742 spores could potentially be attributed to changes in the fungal community. In addition, applying NJAU 4742 spores directly increased the relative abundance of Trichoderma. This change allowed the Trichoderma genus to occupy a more ecological niche in the rhizosphere of tea cuttings and enabled Trichoderma to play a direct role in promoting growth. This finding was consistent with previous research showing that Trichoderma biofertilizer could enhance grassland biomass, with the Trichoderma genus displaying a positive correlation with grassland biomass [78].

5. Conclusions

The SSF process using tea residue as the raw material achieved a high sporulation yield of NJAU 4742, reaching up to 1.8 × 109 CFU/g FW. Applying the fermentation product, diluted approximately 200 times, enhanced the biomass of tea cuttings, altered the structure of fungal communities in the rhizosphere, and increased the relative abundance of the Trichoderma genus, proved to be an economically viable method. The utilization of tea residues for NJAU 4742 fermentation is not only feasible but also recommended to produce biological nursery substrates. Future research should focus on obtaining chlamydospore produced by NJAU 4742 and enhancing the rhizosphere colonization of Trichoderma strains in tea cutting production.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14030526/s1, Figure S1. Effects of different treatments on the fungal Chao1 index (A) and Shannon index (B). CK, no NJAU 4742 spores; T1, applied nursery substrate with 1 × 108 CFU/mL FW NJAU 4742 spores; T2, applied nursery substrate with 5 × 107 CFU/mL FW NJAU 4742 spores; T3, applied nursery substrate with 1 × 107 CFU/mL FW NJAU 4742 spores; T4, applied nursery substrate with 5× 106 CFU/mL FW NJAU 4742 spore. The different letters denote significantly differences according to were calculated using Duncan’s multiple range test at p < 0.05. Figure S2. Spearman’s rank correlation coefficient between fresh weight of tea plant cutting seedlings and the relative abundance of top ten fungal genera, except Trichoderma.

Author Contributions

Conceptualization, Z.M. and S.X.; methodology, Z.M., S.X. and X.W.; software, Z.M., S.X. and J.Z.; validation, X.W. and G.B.; formal analysis, Z.M. and H.L.; investigation, S.X.; resources, X.W.; data curation, Z.M. and S.X.; writing—original draft preparation, Z.M.; writing—review and editing, H.L., R.L. and Q.S.; visualization, S.X., J.Z. and G.B.; supervision, H.L., R.L. and Q.S.; project administration, H.L.; funding acquisition, H.L. and R.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key research and development program (2022YFD1900600, 2022YFE0130200, and 2023YFD1901800) and the Jiangsu Agricultural Science and Technology Innovation Fund [CX (23) 3110].

Data Availability Statement

The datasets generated for this study are available on request to the corresponding author.

Acknowledgments

The authors would like to thank Wanping Fang and Xujun Zhu for their support work.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effects of different Trichoderma strains on the growth of tea cuttings. Survival rate (A) bud length (B). CK—no Trichoderma spore; NJAU 4742—applied nursery substrate with 5.0 × 107 CFU/mL FW NJAU 4742 spores; MD30—applied nursery substrate with 5.0 × 107 CFU/mL FW MD30 spores; JS84—applied nursery substrate with 5.0 × 107 CFU/mL FW JS84 spores. Different letters denote significant differences according to Duncan’s multiple range test at p < 0.05. (n = 3). The results of the homogeneity of variance tests for A and B are p = 0.094 > 0.05 and p = 0.786 > 0.05, respectively, consistent with the hypothesis of homogeneity of variance. In addition, the F values of the two groups of analysis are 6.415 and 15.991 respectively, indicating significant differences between different treatments.
Figure 1. Effects of different Trichoderma strains on the growth of tea cuttings. Survival rate (A) bud length (B). CK—no Trichoderma spore; NJAU 4742—applied nursery substrate with 5.0 × 107 CFU/mL FW NJAU 4742 spores; MD30—applied nursery substrate with 5.0 × 107 CFU/mL FW MD30 spores; JS84—applied nursery substrate with 5.0 × 107 CFU/mL FW JS84 spores. Different letters denote significant differences according to Duncan’s multiple range test at p < 0.05. (n = 3). The results of the homogeneity of variance tests for A and B are p = 0.094 > 0.05 and p = 0.786 > 0.05, respectively, consistent with the hypothesis of homogeneity of variance. In addition, the F values of the two groups of analysis are 6.415 and 15.991 respectively, indicating significant differences between different treatments.
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Figure 2. Effects of different parameters on NJAU 4742 in SSF of tea residue. Tea residue to rice ratio (A), material thickness (cm) (B), inoculum concentration (%) (C), and incubation time (d) (D). Different letters denote significant differences according to Duncan’s multiple range test at p < 0.05. (n = 3). The results of the homogeneity of variance tests for the analysis are PA =0.952 > 0.05, PB = 0.452 > 0.05, PC = 0.586 > 0.05, and PD = 0.494 > 0.05, respectively, consistent with the hypothesis of homogeneity of variance. In addition, the F values of the four groups of analysis are 198, 196, 95, and 373, respectively, indicating significant differences between different treatments.
Figure 2. Effects of different parameters on NJAU 4742 in SSF of tea residue. Tea residue to rice ratio (A), material thickness (cm) (B), inoculum concentration (%) (C), and incubation time (d) (D). Different letters denote significant differences according to Duncan’s multiple range test at p < 0.05. (n = 3). The results of the homogeneity of variance tests for the analysis are PA =0.952 > 0.05, PB = 0.452 > 0.05, PC = 0.586 > 0.05, and PD = 0.494 > 0.05, respectively, consistent with the hypothesis of homogeneity of variance. In addition, the F values of the four groups of analysis are 198, 196, 95, and 373, respectively, indicating significant differences between different treatments.
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Figure 3. Effects of different concentrations of NJAU 4742 spores on the growth of tea cuttings. Fresh weight (g) (A), stem diameter (mm) (B), plant height (cm) (C), and root length (cm) (D). CK—no NJAU 4742 spores; T1—applied nursery substrate with 1.0 × 108 CFU/mL FW NJAU 4742 spores; T2—applied nursery substrate with 5.0 × 107 CFU/mL FW NJAU 4742 spores; T3—applied nursery substrate with 1.0 × 107 CFU/mL FW NJAU 4742 spores; T4, applied nursery substrate with 5.0 × 106 CFU/mL FW NJAU 4742 spores. The different letters denote significant differences according to Duncan’s multiple range test at p < 0.05. (n = 3). The results of the homogeneity of variance tests for the analysis are PA =0.074 > 0.05, PB = 0.013 > 0.05, PC = 0.204 > 0.05, and PD = 0.142 > 0.05, respectively, consistent with the hypothesis of homogeneity of variance. In addition, the F values of the four groups of analysis are 22.7, 17.4, 3.57, and 5.82, respectively, indicating significant differences between different treatments.
Figure 3. Effects of different concentrations of NJAU 4742 spores on the growth of tea cuttings. Fresh weight (g) (A), stem diameter (mm) (B), plant height (cm) (C), and root length (cm) (D). CK—no NJAU 4742 spores; T1—applied nursery substrate with 1.0 × 108 CFU/mL FW NJAU 4742 spores; T2—applied nursery substrate with 5.0 × 107 CFU/mL FW NJAU 4742 spores; T3—applied nursery substrate with 1.0 × 107 CFU/mL FW NJAU 4742 spores; T4, applied nursery substrate with 5.0 × 106 CFU/mL FW NJAU 4742 spores. The different letters denote significant differences according to Duncan’s multiple range test at p < 0.05. (n = 3). The results of the homogeneity of variance tests for the analysis are PA =0.074 > 0.05, PB = 0.013 > 0.05, PC = 0.204 > 0.05, and PD = 0.142 > 0.05, respectively, consistent with the hypothesis of homogeneity of variance. In addition, the F values of the four groups of analysis are 22.7, 17.4, 3.57, and 5.82, respectively, indicating significant differences between different treatments.
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Figure 4. Fungal microbial community compositions of the different treatments. CK—no NJAU 4742 spores; T1—applied nursery substrate with 1.0 × 108 CFU/mL FW NJAU 4742 spores; T2—applied nursery substrate with 5.0 × 107 CFU/mL FW NJAU 4742 spores; T3—applied nursery substrate with 1.0 × 107 CFU/mL FW NJAU 4742 spores; T4—applied nursery substrate with 5.0 × 106 CFU/mL FW NJAU 4742 spores. Differences in the fungal beta diversities were determined via analysis of molecular variance (AMOVA).
Figure 4. Fungal microbial community compositions of the different treatments. CK—no NJAU 4742 spores; T1—applied nursery substrate with 1.0 × 108 CFU/mL FW NJAU 4742 spores; T2—applied nursery substrate with 5.0 × 107 CFU/mL FW NJAU 4742 spores; T3—applied nursery substrate with 1.0 × 107 CFU/mL FW NJAU 4742 spores; T4—applied nursery substrate with 5.0 × 106 CFU/mL FW NJAU 4742 spores. Differences in the fungal beta diversities were determined via analysis of molecular variance (AMOVA).
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Figure 5. Relative abundance of the top ten most abundant fungal genera (A) and Spearman’s rank correlation coefficient of the fresh weight of tea cuttings and Trichoderma (B). CK—no NJAU 4742 spores; T1—applied nursery substrate with 1.0 × 108 CFU/mL FW NJAU 4742 spores; T2—applied nursery substrate with 5.0 × 107 CFU/mL FW NJAU 4742 spores; T3—applied nursery substrate with 1.0 × 107 CFU/mL FW NJAU 4742 spores; T4—applied nursery substrate with 5.0 × 106 CFU/mL FW NJAU 4742 spores.
Figure 5. Relative abundance of the top ten most abundant fungal genera (A) and Spearman’s rank correlation coefficient of the fresh weight of tea cuttings and Trichoderma (B). CK—no NJAU 4742 spores; T1—applied nursery substrate with 1.0 × 108 CFU/mL FW NJAU 4742 spores; T2—applied nursery substrate with 5.0 × 107 CFU/mL FW NJAU 4742 spores; T3—applied nursery substrate with 1.0 × 107 CFU/mL FW NJAU 4742 spores; T4—applied nursery substrate with 5.0 × 106 CFU/mL FW NJAU 4742 spores.
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Meng, Z.; Xiang, S.; Wang, X.; Zhang, J.; Bai, G.; Liu, H.; Li, R.; Shen, Q. Turning Waste into Wealth: Utilizing Trichoderma’s Solid-State Fermentation to Recycle Tea Residue for Tea Cutting Production. Agronomy 2024, 14, 526. https://doi.org/10.3390/agronomy14030526

AMA Style

Meng Z, Xiang S, Wang X, Zhang J, Bai G, Liu H, Li R, Shen Q. Turning Waste into Wealth: Utilizing Trichoderma’s Solid-State Fermentation to Recycle Tea Residue for Tea Cutting Production. Agronomy. 2024; 14(3):526. https://doi.org/10.3390/agronomy14030526

Chicago/Turabian Style

Meng, Zhen, Shuangshuang Xiang, Xue Wang, Jian Zhang, Guoxin Bai, Hongjun Liu, Rong Li, and Qirong Shen. 2024. "Turning Waste into Wealth: Utilizing Trichoderma’s Solid-State Fermentation to Recycle Tea Residue for Tea Cutting Production" Agronomy 14, no. 3: 526. https://doi.org/10.3390/agronomy14030526

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