Efficacy of Trichoderma asperellum TC01 against anthracnose and growth promotion of Camellia sinensis seedlings

Journal Pre-proofs Efficacy of Trichoderma asperellum TC01 against anthracnose and growth promotion of Camellia sinensis seedlings Jing Shang, Bingliang Liu, Ze Xu PII: DOI: Reference:

S1049-9644(19)30706-6 https://doi.org/10.1016/j.biocontrol.2020.104205 YBCON 104205

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Biological Control

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Please cite this article as: Shang, J., Liu, B., Xu, Z., Efficacy of Trichoderma asperellum TC01 against anthracnose and growth promotion of Camellia sinensis seedlings, Biological Control (2020), doi: https://doi.org/10.1016/ j.biocontrol.2020.104205

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Efficacy of Trichoderma asperellum TC01 against anthracnose and growth promotion of Camellia sinensis seedlings

Jing Shang1†*, Bingliang Liu2†, Ze Xu1 Tea Research Institute of Chongqing Academy of Agricultural Science, Chongqing 402160, China, 1

Business College of Ludong University, Yantai 264025, China, 2 †

First and second authors contributed equally to this work and should be considered co-first 

authors. Email address: [email protected] (Bingliang Liu); [email protected] (Ze Xu) * Corresponding author: Jing Shang Mailing address: [email protected] Address: Tea Research Institute, Chongqing Academy of Agricultural Science，No. 2, Gui Shan Road, Yongchuan District, Chongqing 402160, China

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Abstract Tea plant (Camellia sinensis) is a perennial evergreen woody plant that is economic importance worldwide. Its health functions and distinctive taste depend on the production of secondary metabolites, which are affected by biotic and abiotic stresses. Biological control is considered a feasible alternative to chemical control as well as an eco-friendly method for controlling pathogens. In this study, we investigated the role of Trichoderma asperellum TC01 in the regulation of C. sinensis growth and the activation of defence responses against Colletotrichum gloeosporioides C62 by developing a biocontrol–plant–pathogen interaction system. When C. sinensis was co-cultivated with T. asperellum TC01, it was able to reduce 58.37% of disease severity compared with other treatments. Furthermore, compared with control plant growth, T. asperellum TC01-treated plants showed an increase in shoot height (7.5%), stem diameter (34.09%), shoot fresh weight (81.18%), root fresh weight (93.75%), shoot dry weight (85.71%) and root dry weight (115.38%) at 45 days after inoculation under greenhouse conditions. RNA sequencing analysis revealed that 48 hours after inoculation, key genes were up-regulated in the flavonoid biosynthesis pathway of TC01-treated plants (T4) and down-regulated in leaves of C. gloeosporioides C62-treated plants compared with that of the control plants, indicating that TC01 triggers flavonoid and phenylpropanoid pathways during the early stages of the interaction with C. sinensis. Furthermore, higher expression levels of genes associated with the jasmonic acid and ethylene signalling pathway in the roots of TC01- and C62-treated plants (T3) than in either TC01- or C62-treated plants, indicated that TC01 triggered the induced systemic response in C. sinensis. Genes involved in the flavonoid, phenylpropanoid, jasmonic acid and ethylene play an important role in the resisitance against anthracnose. These results indicate induction of defense

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mechanisms by TC01 is potentially a powerful approach for controlling Camellia sinensis disease, which may provide a management opportunity for the sustainable use of TC01 treatments in tea plantations. KEYWORDS: Tea plantation, Trichoderma asperellum TC01, Colletotrichum gloeosporioides C62, Plant defense, Tourism disturbance areas 1. Introduction Tea (Camellia sinensis (L.) O. Kuntze) is a tree species of economic importance worldwide. Its health functions and distinctive taste are due to the production of secondary metabolites, such as flavonoids (or phenolic compounds), theanine and alkaloids, which are affected by soil quality and biotic and abiotic stresses (Chen et al., 2008; Li et al., 2015). To meet the expected demand for tea and to control disease, plants are sprayed with chemical fungicides between five and seven times a year, which is laborious and expensive. Furthermore, the extensive use of chemical control methods contributes to the emergence of fungicide-resistant pathogens (Thonar et al., 2017), which may negatively impact the indigenous microbial community by disturbing the natural distribution of microbes in the soil, as well as diverse pollution issues and the degradation of ecosystems, with negative effects on humans (Wang et al., 2016; Liu, 2013). In addition, there is also a risk that tourists attracted to tea gardens in recent years by the development of recreational activities may accidentally ingest fungicides. The trampling of the ground by these tourists may dramatically affect the soil and rhizosphere ecology of tea gardens. The proportion of C. sinensis cultivated without the application of chemical fungicides or with fewer applications of chemical fungicide has increased in recent years because of the huge

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impact on tea productivity worldwide. Thus, novel and eco-friendly aspects of agricultural practices, and sustainable ways of managing disease control against plants have gained considerable attention in recent years (Kejela et al., 2016; Li et al., 2018b; Muhammad et al. 2018; Wu et al., 2017). Plant growth-promoting microorganisms (PGPMs) have proved to be powerful tools in plant health management (Hou et al., 2018; Huang et al., 2015; Nassal et al., 2017; Berendsen et al., 2018). It offered excellent opportunities as potential biofertilizers and biopesticides (Pagnani et al., 2018; Palmieri et al., 2016), and can modify plant responses to biotic and abiotic stresses (Barnawal et al., 2017; Shameer and Prasad, 2018). It is known that PGPMs use distinct mechanisms to enhance plant growth while suppressing disease caused by its rival (Santoro et al., 2015; Singh et al., 2016, 2018), have the potential to replace chemical fertilizers and pesticides in agriculture (Muhammad et al., 2017; Myresiotis et al., 2015; Nkebiwe et al., 2016). Furthermore, the mechanisms of PGPMs mainly include the induction of host plant-produced siderophores, β-1,3-glucanase, chitinase, antibiotics, metabolites and cyanide to confine pathogen spread (Chandran et al., 2005; Bano and Muqarab, 2017; Gouda et al., 2018), the induction of the plant defence system (Harman et al., 2004; Figueredo et al., 2017; Guijarro et al., 2018) to diminish plant disease symptoms at the root and foliar levels (Haney et al., 2017; Li et al., 2018b), the improvement of soil nutrients, nitrogen fixation, the alteration of plant metabolic composition (Trinh et al., 2018; Zhang et al., 2019), and pesticide degradation (Roy et al., 2018). Trichoderma species are soil-living fungi that are used worldwide as biocontrol and plant growth promoting agents (Kumar, et al. 2012; Lopez-Coria et al., 2016). Trichoderma spp. have been reported to exhibit biostimulating abilities when used to treat a range of vegetables, maize

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and soybean, inducing plant-resistance mechanisms, root development and plant growth (Segarra, et al., 2010; Chinheya et al., 2017; Fu et al., 2018; Jogaiah et al., 2018). Early studies on Trichoderma mostly focused on their use as potential biocontrol agents. However, as our understanding of Trichoderma has deepened, research into the mechanism associated with promoting plant growth and vigour has gradually increased. To deeper understanding mechanisms of the tripartite interaction biocontrol-plant-pathogen (Trichoderma spp.–C. gloeosporioides–C. sinensis), the objectives of this study were, (ⅰ) to identify and characterize Trichoderma spp. isolated from the C. sinensis rhizosphere, (ⅱ) to evaluate the antagonistic activity of selected Trichoderma spp. isolates against C. gloeosporioides in vitro, (ⅲ) to determine the efficacy of Trichoderma spp. isolates to inhibit C. gloeosporioides disease development and to promote plant growth, (ⅳ) to evaluate the defence mechanisms of the Camellia sinensis cv. Fuding Dabaicha against the pathogen when pre-treated with the selected Trichoderma spp. isolate. 2. Materials and methods 2.1. Isolation and identification of anthracnose Thirty C. gloeosporioides-infected leaves were collected from Camellia sinensis cv. Fuding Dabaicha plants growing in plantations with recreational stress caused by tourists visiting during 2017 in the Yongchuan district of Chongqing in China. The leaves were collected in sterile plastic zip-lock bags, transported to the laboratory under environmental temperature and stored at 4 °C before further processing. Isolates were isolated by a single spore isolation technique as described by Cai, et al (2009). For pathogenicity tests, wound inoculation of plants was performed in vitro based on the method described by Wang et al (2016). The pathogen was identified through

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morphological and molecular characterization by amplifing of the ITS region of ribosomal DNA (rDNA) using the universal primers ITS4 and ITS5. 2.2. Isolation of Trichoderma spp. Thirty Soil samples were collected from Camellia sinensis cv. Fuding Dabaicha plants growing in plantations with recreational stress during 2017 to 2018 in the Yongchuan district of Chongqing in China. Rhizospheric soils and root zone soils were collected according to the method described by Bulgarelli et al. (2012). The plant roots soils were collected in sterile plastic zip-lock bags, transported to the laboratory under environmental temperature and stored at 4 °C before further processing. The cultures were isolated with Trichoderma selective medium through serial dilutions (from 10−1 to 10−5) at 10-5 dilution (Davet and Rouxel, 2000). The emergent colonies, tentatively identified as Trichoderma were pure cultured on to PDA. Conidia of all isolated Trichoderma strains were suspended in glycerol (15% v/v) and stored at −80°C for long-term storage. 2.3. In vitro screening of antagonistic Trichoderma spp. A dual culture technique (Castillo et al., 2011) was used to assess the antagonistic effect of all Trichoderma isolates against C. gloeosporioides C62 by placing 5-mm diameter fungal plugs of a Trichoderma isolate and C. gloeosporioides C62 5 cm apart on Petri dishes (90 mm diameter) containing PDA. Plates were then incubated for 10 days at 28 ± 2°C in the dark. The experiment was performed in a completely randomized design with four replications and each experiment was repeated twice. Percent inhibition of the test pathogen by Trichoderma spp. was calculated by recording the radial growth of mycelium of the pathogen (Campanile et al., 2007). 2.4. Identification of Trichoderma isolate TC01

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Hyphal morphology and colony growth patterns of 7-day-old cultures of Trichoderma isolate TC01 grown on PDA at 28°C in the dark were examined under a light microscope (Leica DM750) and compared with published descriptions (Wang et al., 2016). Isolates were grown on PDA at 28°C for 10 days in the dark before scraping the mycelium from the agar surface with a sterile blade. DNA was extracted using a genomic DNA kit (Ezup Column Fungi Genomic DNA Purification Kit, Sangon Biotech). The concentration of the genomic DNA obtained was determined using a spectrophotometer at 260 nm. Trichoderma isolates were identified through molecular characterization by amplifing of the ITS region of ribosomal DNA (rDNA) using the universal primers ITS4 and ITS5, translation elongation factor 1 alpha (tef1a) gene using the universal primers EF728 and EF1. Sequencing were performed by Sangon Biotech, Shanghai, China. Sequences generated for the Trichoderma isolates obtained in this study were compared with reference sequences of other closely related species, including some undescribed taxa, obtained from GenBank (https://blast.ncbi.nlm.nih.gov). A multiple sequence alignment was created using the CLUSTAL-X program to compare the sequence data from Trichoderma-like isolates with the corresponding sequences of several species of Trichoderma. Phylogenetic analyses were performed using the neighbour-joining method and the MEGA 7.0 program, with statistical validity tested via bootstrap analysis (1,000 replicates). 2.5. Efficacy of T. asperellum TC01 on management of anthracnose and growth promotion of Camellia sinensis seedlings under greenhouse conditions 2.5.1 Biocontrol efficacy of T. asperellum TC01 against C. gloeosporioides C62 All Ca sinensis cv. Fuding Dabaicha seedlings used in this study were obtained from one-year

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cuttings. The seedlings were grown in pots (8 cm in diameter and 7 cm in height) containing 250 g of sterilized quartz sand (irrigated with Hoagland nutrient solution), with two seedlings per pot. The seedlings were grown in a greenhouse with the following conditions 25°C and 85% relative humidity. All seedlings used in the experiments were two weeks old at the start of the experiment. To assess the biocontrol efficacy of TC01 against C62 under greenhouse conditions, C. sinensis seedlings were subjected to the following treatments: (T1) plants without TC01 or C62 (healthy control); (T2) C62-inoculated plants (disease control); (T3) C62- and TC01-inoculated plants and (T4) TC01-inoculated plants. The plants were grown in a 28°C phytotron under a 10-h light/14-h dark cycle with a light intensity of 150–200 μmol m−2 s−1 and 60–70% humidity for 45 days. T. asperellum TC01 conidial suspension containing 3 × 105 conidia/ml were also mixed with the sterilized quartz sand at a ratio of 1:9 (w:w). Seedling leaves were challenge-inoculated with a C62 conidial suspension containing 1 × 105 conidia/ml. Control plants were treated with sterile distilled water. The efficacy of TC01 in suppressing disease was evaluated by visually assessing lesions that developed on leaves. The disease rating was scored on a scale of 0 to 5, as described in Table 1. [Table 1] A completely randomized design was used with a total of fourty seedings (four treatment × ten replicates), and the experiment was carried out twice. The disease severity was calculated for every replication by determining the disease index: 100 × ∑(n × r)/(N × maximum value) (Madden et al., 2007), where r is the rating value (0–5) and represents the proportion of the total leaf area with lesions; n is the number of infected leaves with

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a rating of r, and N is the total number of leaves tested for each replication (each pot). Disease evaluations were made for each replication 2, 6, 10, 14, 18, 22, 26 and 30 days after inoculation (DAI). The disease severity values were used to plot the disease progress curve. The area under the disease progress curve was utilized to assess the severity of the disease intensity over time and to determine the effectiveness of the TC01 treatment. The area under disease progress curve (AUDPC) was utilized to assess the severity of the disease intensity over time and to determine the effectiveness of TC01 treatment. AUDPC was measured using the equation:

N= the total number of observations; yi = percent disease severity data collected at various times (ti).

2.5.2. Assessment of C. sinensis agronomic traits after TC01 and/or C62 treatment After harvesting, seedings (samples from 2.5.1) was measured to determine the height of the plant (from the soil surface to the top of the highest panicle of each plant), the stem diameter, and the shoot and root fresh weight. The samples were fixed in an oven at 105°C for 30 min, and then dried at 80°C for 48 h or until there was a constant mass, and dry weight was recorded. Analysis of variance (ANOVA) was conducted using SPSS 19 to determine significant differences among the agronomic traits assessed. A least significant difference test was performed to compare the means. 2.5.3. Global analysis of gene expression during the early stages of the C. sinensis–TC01–C62 interaction based on RNA-seq The treatments used in this experiment were as described in section 2.5.1, forty-eight hours after inoculation, a bud with two leaves and a root tip were collected, snap-frozen in liquid nitrogen and stored at –80°C for later use, all the experiments were carried out in triplicate. 9

Total RNA was extracted using the RNeasy Plus Mini kit (Qiagen, Valencia, CA, USA), and the RNA integrity was confirmed using RNA 6000 Nano LabChips with a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Qualified total RNA was further purified using an RNAClean XP Kit (Beckman Coulter, Kraemer BoulevardBrea, CA, USA) and an RNase-Free DNase Set (Qiagen, Hilden, Germany). Then, a cDNA library was built. Solexa sequencing of paired-ends (2 × 100) was performed, which produced at least 100 million reads per sample. The transcriptome data were subjected to conventional analyses, including data pre-processing, genomic mapping, gene expression analysis, transcript expression analysis, alternative splicing analysis, analysis of differentially expressed genes, and GO/KEGG enrichment analysis of differentially expressed genes and non-differentially expressed genes (Trapnell et al., 2009, 2010; Kanehisa et al., 2012). Genes related to plant hormone signal transduction and phenylpropanoid biosynthesis were analysed in roots and genes related to flavonoid biosynthesis were analysed in leaves and compared to public genomic data. All homologous genes were assembled and matched to RNA-seq data to determine their expression levels. 3. Results 3.1. Identification of the C. sinensis pathogen The identity of the C. sinensis pathogen (isolate C62) was confirmed to be Colletotrichum gloeosporioides based on morphological observations (Fig. 1) and sequence analysis of the ITS region and a BLAST search with 100% identity to another reference C. gloeosporioides strain available in GenBank (GenBank MN735438). C. sinensis seedlings inoculated with C. gloeosporioides C62 developed lesions that were similar to those observed on the diseased

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samples collected from the tea garden. Isolate C62 was reisolated from these lesions, thereby fulfilling Koch’s postulates. [Fig 1] 3.2. Isolation of Trichoderma spp. and their antagonistic activities We obtained 45 Trichoderma isolates from rhizospheric and root zone soils. Of the 45 Trichoderma spp. isolated, 14 isolates showed antifungal activity towards C62 mycelia when grown in dual cultures. TC01 exhibited the strongest antagonistic activity of the isolates tested, with a PIRG of 90% (Fig. 2a, b) and, microscopy observations revealed that TC01 hyphae coiled around and penetrated the C62 hyphae (Fig. 2c). Therefore, isolate TC01 was chosen for use in subsequent experiments. [Fig. 2] 3.3. Colony morphology, molecular identification and phylogenetic tree Morphological observations revealed that after five days of culture, the upper side of the TC01 colony was dark green (spore colour) and the underside was white (Fig. 3). The morphology of the mycelium was coarse and dark-green spores began to form in the centre of the colony at high rates (Fig. 3). Based on preliminary morphological observations of the conidiophores and spores, the strain was confirmed to be Trichoderma asperellum and named it TC01. [Fig. 3]

The ITS and translation elongation factor 1 alpha (tef1a) genes sequences of isolate TC01 were deposited in GenBank under accession number MH752042 and MN813963.A BLAST search revealed that the sequences were similar to that of other Trichoderma species. The phylogenetic tree derived from the ITS and tef1a gene sequences of TC01 and Trichoderma

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species obtained from NCBI GenBank is shown in Fig. 4 and Fig. 5. TC01 and T. asperellum were clustered closely together. Based on morphological and sequencing analyses, we identified the TC01 isolate as a Trichoderma asperellum strain and named it T. asperellum TC01, it has been deposited in the Chinese General Microbiological Collection Centre (Beijing, China) as strain CGMCC No.3.19218. [Fig. 4] [Fig. 5] 3.4. Biocontrol efficacy of T. asperellum TC01 under greenhouse conditions The initial disease symptoms appeared 3 DAI. Typical symptoms observed on leaves were lesions with a grey or white centre with black dots in the lesion. Symptoms increased daily from 3 DAI and peaked 26 DAI in pathogen-inoculated plants (T2), with the disease severity of T2 plants rising from 20 to 73 between 6 and 30 DAI. By contrast, the disease severity of T3 plants (pathogen- and TC01-inoculated plants) rose from 6 to 25 during the same period. No disease development was observed in either T1 (uninoculated) or T4 (TC01-inoculated) plants (Fig. 6). As compared to T2 plants, T3 plants had a significantly lower AUDPC (487 units) (P<0.05) with 58.37% (P<0.05) of disease reduction. [Fig. 6] 3.5. Effect of inoculation treatments on plant growth Forty-five DAI, plants were harvested to assess plant growth. Plants inoculated with T. asperellum TC01 showed enhanced growth (P<0.05), especially the roots (Fig. 7). The mean shoot height, stem diameter, fresh and dry shoot weight, and fresh and dry root weight of T4

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(TC01-inoculated) plants were significantly enhanced (P<0.05) compared with those of T1 (control), T2 (pathogen-inoculated) and T3 (pathogen and TC01-inoculated) plants. At 45 DAI, the shoot height, stem diameter, shoot fresh weight, root fresh weight, shoot dry weight, and root dry weight of T4 plants was 7.5%, 34.09%, 81.18%, 93.75%, 85.71% and 115.38% greater, respectively, than that of T1 control plants when grown under greenhouse conditions. Furthermore, mean shoot and root fresh weights, and the root dry weight of T3 plants were significantly higher (P<0.05) than those of T1 (control) plants. In addition, the shoot fresh weight of T4 (1.83 g ± 0.02 g) and T3 (1.42 g ± 0.1 g) plants was significantly higher than that of T2 plants (0.79 g ± 0.04 g). The lowest levels of growth were recorded for T2 plants (Table 2). [Fig. 7] [Table 2] 3.6. Effect of T. asperellum TC01 on the flavonoid biosynthesis pathway of C. sinensis leaves during the early stages of the C. sinensis–TC01–C62 interaction, determined by RNA-seq We assessed whether the expression levels of genes associated with the flavonoid biosynthesis pathway in leaves were changed during the tripartite interaction (C. sinensis–TC01–C62), including PAL, C4H, 4CL, CHS, CHI, F3’5’H, F3’H, DFR, ANS, LAR and LDOX. RNA-seq data showed that all the genes that showed a significant change in expression levels in T4 plants were up-regulated, and most of the genes in T2 and T3 plants that showed a significant change in expression levels were down-regulated (Fig. 8; Table S1). [Fig. 8] 3.7. Effect of T. asperellum TC01 on the plant hormone signal transduction pathway in roots during the early stages of the C. sinensis–TC01–C62 interaction, determined by RNA-seq

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We assessed whether differences in the plant hormone signal transduction pathway in roots had an impact on the responsive gene expression level in the tripartite interaction (C. sinensis–TC01–C62). In root tissue, higher expression levels were detected in T2 and T3 plants than in T4 plants. The expression of all the indole-3-aceteic acid (IAA), jasmonic acid (JA) and ethylene (ET) genes was up-regulated. These results demonstrate that inoculating the rhizosphere of C. sinensis with T. asperellum TC01 could enhance the expression of disease resistance genes (Fig. 9; Table S2). The ISR was elicited through the JA-regulated priming mechanism and ET-dependent signalling pathway. T. asperellum TC01 induced changes in the secondary metabolic pathways of C. sinensis that were related to plant resistance in roots. Most transcripts were related to genes encoding enzymes involved in the biosynthesis of alkaloids, flavonoids, terpenoids and amino acids, especially genes encoding the phenylalanine pathway enzymes. The transcript levels of genes encoding phenylalanine

ammonia-lyase

(PAL),

4-coumarate-CoA

ligase

(4CL3)

and

caffeoyl-CoA-O-methyltransferase (ATOMT) were up-regulated to various degrees, with higher expression levels detected in T2 and T3 than in T4 plants (Fig. 10; Table S3). [Fig. 9] [Fig. 10] 4. Discussion Studies have shown that the inoculation of plants with PGPMs can change the gene expression profile of plants, affecting genes involved in metabolism, signal transduction and stress responses. One of the reported mechanisms of plant growth promotion by PGPM strains is the production of hormones such as IAA, cytokinins and gibberellins to stimulate plant growth.

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Cytokinin and auxin pathway genes were upregulated in plants inoculated with TC01 in this study, and after 45 days there was a significant increase in fresh and dry weight and lateral root length in treated seedlings, suggesting that T. asperellum TC01 can affect the growth and dry matter accumulation. These results indicate that the plant growth-promoting activity might be caused by the production of growth-inducing hormones. In future studies, we will test whether the growth-promoting abilities of TC01 that were observed in plants grown under greenhouse conditions in soil are also present when plants are grown under field conditions. Another reported effect of treating plants with Trichoderma spp. is the induction of ISR. Several Trichoderma spp. have been reported to induce systemic resistance (Baiyee et al., 2019; Singh et al., 2016), triggering defences such as high phenylpropanoid activities and lignification against phytopathogens (Singh et al., 2016; Ben Amira et al., 2017). In this study, the disease progression rate in plants pre-treated with T. asperellum TC01 was slower than that in pathogen-infected plants. Furthermore, RNA-seq results showed that the expression of genes associated with various phytohormone-related pathways were up-regulated in T2, T3 and T4 plant roots, including responses to JA and ethylene. Meanwhile, expression levels of defence-related genes in T3 plants were higher than those in T2 plants, suggesting that T. asperellum TC01 rapidly colonized the rhizosphere and induced the expression of resistance genes within 48 h of inoculation. Co-inoculating T. asperellum TC01 and C. gloeosporioides C62, up-regulated the expression of phenylpropanoid biosynthesis-related genes, including PAL and 4CL. This suggests that the defence response of TC01-treated C. sinensis plants to C62 infection might be enhanced owing to the synthesis of lignans, flavonoids and alkaloids through phenylpropane metabolism. These results are likely to be related to the ability of T. asperellum TC01 to induce host ISR

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through the JA- and ET-regulated priming mechanism in C. sinensis samples. We also suggest that plant hormone signal transduction pathway genes are involved in the regulation of plant hormones to activate plant defence responses. Previous studies had shown that plants produce and accumulate a large number of secondary metabolites in response to many biological and abiotic stresses, such as infections by pathogenic bacteria, pest feeding, mechanical injury, and exogenous plant hormones (Yedidia et al., 2003). In this study, T. asperellum TC01 altered flavonoid and phenylpropanoid biosynthesis pathway-related genes: PAL, C4H, 4CL, CHS, CHI, F3’5’H, F3’H, DFR, ANS, LAR and LDOX. In T4 plants, these genes were up-regulated. In T2 and T3 plants, fewer genes were expressed and these were down-regulated. These results suggest that C62 may induce the down-regulation of flavonoid biosynthesis pathway-related genes, which may affect the formation of flavonoids in leaves, whereas TC01 might induce the synthesis of a group of flavonoids, and synthesize lignin to reinforce cell walls or produce phenolic compounds to improve resistance to pathogen attack. These findings suggest that plant secondary metabolism, especially flavonoid metabolism, plays an important role in the induction of disease resistance in tea leaves by T. asperellum TC01 at the level of overall gene transcription. High concentrations of flavan-3-ols (aka catechins) and anthocyanins are present in tea plants with purple leaves, which are synthesized through the flavonoid metabolic pathway, and have beneficial effects on both plant and human health (Li et al., 2015). A previous study has shown that there is a high correlation between the expression levels of related genes (except LAR and F3’H) and the concentration of total catechins in green leaves (Zhou et al., 2016), so one of our next steps will be to examine the relationship between TC01 and total catechins.

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The up-regulation of genes expressed in T. asperellum TC01-treated plants indicates that the promotion of plant growth may involve a combination of mechanisms, such as the induction of ISR, the production of hormones and the induction of secondary metabolites. These findings are consistent with previous studies in which vegetable crops (e.g., tomato, brinjal, chilli, okra, ridge gourd and guar) were inoculated with biocontrol Trichoderma spp (Singh et al., 2016). This suggests that the promotion of plant growth by Trichoderma may involve the induction of common pathways. In recent years, China's many tea plantation have begun to be developed to encourage visitors and recreation, which may further increase stress on tea plantation. Field investigations have found that the incidence of anthracnose disease on tea plantation was significantly higher in recreational areas than in non-recreational areas (Shang et al., unpublished results). This may be because, on the one hand, tea tree branches can become damaged by recreational activities, exacerbating the infection and spread of disease; on the other hand, the trampling of the ground by tourists may change the physical and chemical attributes of the soil and the microbial  environment around the roots, reducing soil nutrients, available nutrients, the soil invertase, urease, acid phosphatase activity and microbial activity (Zhu and Bai, 2015). The T. asperellum TC01 may provide a new approach to manage tea plantation that are increasingly subjected to recreational disturbance stresses. 5. Conclusion The results obtained in this study demonstrate the potential of using T. asperellum TC01 as a biocontrol agent against the pathogenic fungus C. gloeosporioides C62 and as a plant growth promoter of C. sinensis. T. asperellum TC01 significantly increased plant growth attributes and

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demonstrated up-regulation of defence-related genes in both infected and non-infected plants. TC01 up-regulated key genes in the flavonoid biosynthesis pathway of tea plants. Hence, we conclude that T. asperellum TC01 is a potential biocontrol and bioenhancer candidate for disease management and growth promotion of C. sinensis, serving as an ideal alternative to conventional fertilizers and pesticides to improve crop productivity in an ecologically stable and sustainable manner. Elicitation of defense mechanisms by T. asperellum TC01 is potentially a powerful approach for controlling plant diseases and represents an alternative strategy to environmentally undesirable chemical-based methods. Acknowledgements The authors thank teacher Shang-jun Huang for supporting the Ca. sinensis seedlings. This research was supported by the funds of General Fundamental and Advanced Research Projects of Chongqing Science & Technology Commission (cstc2018jcyjAX0586), Performance Incentive Guidance for Scientific Research Institution of Chongqing (cstc2018jxjl80015), General Fundamental and Advanced Research Projects of Yongchuan Chongqing (Ycstc,2018nb0104). References Baiyee, B., Pornsuriya, C., Ito, S. i., and Sunpapao, A., 2019. Trichoderma spirale T76-1 displays biocontrol activity against leaf spot on lettuce (Lactuca sativa L.) caused by Corynespora cassiicola or Curvularia aeria. Biological Control. 129, 195-200. Bano, A., and Muqarab, R., 2017. Plant defence induced by PGPR against Spodoptera litura in tomato (Solanum lycopersicum L.). Plant Biol (Stuttg). 19(3), 406-412. Barnawal, D., Bharti, N., Pandey, S.S., Pandey, A., Chanotiya, C. S., and Kalra, A., 2017. Plant growth-promoting rhizobacteria enhance wheat salt and drought stress tolerance by altering

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sterile PDA plug (top) and C62 (bottom); (b) TC01 (top) and C62 (bottom); (c) TC01 hyphae coiling around and penetrating C62 hyphae. Fig. 3. Morphological structures of Trichoderma asperellum TC01. a. Colony morphology after 5 d growth at 25°C on PDA; b. spores; c. conidiophores. Fig.4.Neighbour-joining tree showing the evolutionary relationships among different Trichoderma species based on their rDNA ITS sequences. Protocrea pallida CBS 299.78 was used as an outgroup. Fig.5.Neighbour-joining tree showing the evolutionary relationships among different Trichoderma species based on translation elongation factor 1 alpha (tef1a) gene sequences. Beauveria brongniartii isolate 3258 was used as an outgroup. Fig. 6. Disease severity over time after the application of treatments. T1 = Camellia sinensis plants that were not inoculated with either the pathogen or Trichoderma asperellum TC01 (control), T2 = pathogen-inoculated plants, T3 = pathogen- and TC01-inoculated plants, and T4 = TC01-inoculated plants. Fig. 7. One-year-old Camellia sinensis cutting seedlings 45 DAI. a.T1 (control) plants; b. T2 (pathogen-inoculated) plants; c. T3 (pathogen- and Trichoderma asperellum TC01-inoculated) plants; and d. T4 (TC01-inoculated) plants. Bar = 5cm Fig. 8. Heatmap of flavonoid biosynthesis pathway genes expressed in Camellia sinensis leaves of T2 (Colletotrichum gloeosporioides C62-inoculated), T3 (C62- and Trichoderma asperellum TC01-inoculated) and T4 (TC01-inoculated) plants, based on RNA-seq.

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Fig. 9. Heatmap of plant hormone signal transduction pathway genes expressed in Camellia sinensis roots of T2 (Colletotrichum gloeosporioides C62-inoculated), T3 (C62- and Trichoderma asperellum TC01-inoculated) and T4 (TC01-inoculated) plants based on RNA-seq. Fig. 10. Heatmap of phenylalanine pathway genes expressed in Camellia sinensis roots of T2 (Colletotrichum gloeosporioides C62-inoculated), T3 (C62- and Trichoderma asperellum TC01-inoculated) and T4 (TC01-inoculated) plants based on RNA-seq.

Author contributions statement Jing Shang led the program, designed and participated to all of the experiments, and co-wrote the first draft of the article. Bing-liang Liu participated in the analysis of data, co-wrote the first draft of the article and edited. Ze Xu gave some advise during the experiments.

Highlights:   

Trichoderma asperellum TC01, a potential biocontrol agent in Camellia sinensis. TC01 reduced disease severity, while maintaining C. sinensis seedlings growth. TC01 up-regulated defence-related genes.

Table 1 Disease scale used for rating Colletotrichum gloeosporioides C62 disease intensity Code

Predominant lesion type

28

0

No evidence of lesions

1

Pin-point-sized brown specks or larger brown specks with no sporulation centre

2

Round necrotic grey spots (0.5–1.5 mm in diameter) with a distinct brown margin (less than 10% of leaf area infected)

3

Leaf area covered with typical lesions 1.5–4.0 mm in diameter (an area of 11%-50% of leave area infected)

4

Leaf area covered with typical lesions 4–7 mm in diameter (an area of 51%-75% of leave area infected)

5

Leaf area covered with typical lesions more than 7 mm in diameter (more than 75% of leave area infected)

Table 2 Effect of Trichoderma asperellum TC01 on Camellia sinensis seedling growth Treatmen

Shoot height

Stem diameter Fresh shoot (g)

Fresh root (g)

Dry shoot (g)

Dry root (g)

t

(cm)

(cm)

T1

16.38 ± 0.3b

0.44 ± 0.04b

1.01 ± 0.02c

0.48 ± 0.02c

0.39 ± 0.01b

0.13 ± 0.02c

T2

15.52 ± 0.4c

0.39 ± 0.1c

0.79 ± 0.04d

0.45 ± 0.02c

0.31 ± 0.02b

0.12 ± 0.04c

T3

16.58 ± 0.3b

0.48 ± 0.2b

1.42 ± 0.1b

0.79 ± 0.03b

0.38 ± 0.03b

0.2 ± 0.02b

T4

17.61 ± 0.4a

0.59 ± 0.03a

1.83 ± 0.02a

0.93 ± 0.05a

0.78 ± 0.04a

0.28 ± 0.03a

T1, plants that were not inoculated with either Trichoderma asperellum TC01 or Colletotrichum gloeosporioides C62 (healthy control); T2, C62-inoculated plants (disease control); T3, C62- and TC01-inoculated plants; and T4, TC01-inoculated plants. Data shown are mean values ± the XX.

29

Mean values followed by different lowercase letters are significantly different from each other (P<0.05).

30

31

32

33

34

35