Institute of Plant and Environment Protection,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,P.R.China

Abstract Plant growth promoting fungi are receiving increased attention as valuable beneficial microorganisms in crop cultivation due to their capacity to produce bioactive substances,promote plant growth and enhance immune defense functions.In this study,a novel Trichoderma isolate,designated as TM2-4,was screened from healthy tomato rhizosphere soil and identified as Trichoderma afroharzianum.Culture filtrate of the isolate TM2-4 displayed obvious bioactive substance production and an evident effect in promoting tomato seed germination,with hypocotyl length,radical length and vigor index increased by 28.7,19.4 and 62.1%,respectively,after a 100-fold dilution treatment.To assess the promotion effect and related mechanism of isolate TM2-4,the plant biological indexes and gene expression profiles of tomato plants treated with or without T.afroharzianum TM2-4 microbial agent were investigated by greenhouse pot experiment and RNA sequencing.The results demonstrated that T.afroharzianum TM2-4 significantly promoted tomato plant growth in terms of plant height,dry weight,number of leaves per plant and root activity,through efficient colonization in the rhizosphere and root system of the plants.Transcriptome analyses identified a total of 984 differentially expressed genes in T.afroharzianum microbial agent inoculated tomato roots,which were mainly engaged in the biological process of phytohormone homeostasis,antioxidant activity,as well as metabolic pathways including phenylpropanoid biosynthesis and glutathione metabolism.These findings provide useful information for understanding the mechanism of isolate TM2-4 for tomato plant growth promotion,which would facilitate further development of T.afroharzianum TM2-4 microbial agent for use in vegetable crop production.

Keywords:Trichoderma afroharzianum,Solanum lycopersicum L.,plant growth promotion,transcriptome analyses,qRTPCR

1.Introduction

Tomato (Solanum lycopersicumL.) constitutes one of the most important vegetable crops in the world,the fruits of which are a rich source of antioxidants including vitamins,phenolics,flavonoids and folic acid (Hernández-Herreraet al.2014;Perveenet al.2015).To meet the demands of international markets,tomato growers use excessive amounts of chemical fertilizers to ensure high yields,which are sometimes hazardous to human health and the environment (Mengistieet al.2017).The growing concern regarding intensive use of chemical fertilizers has resulted in an increasing attention towards more eco-friendly planting strategies (Wanget al.2016).Proper use of beneficial microbial agents represents a viable strategy to maintain the sustainable and healthy development of tomato production(Caiet al.2015).

Plant growth promoting microbes are widely accepted as offering an environmentally-friendly green approach in agricultural cultivation,due to their ability to stimulate plant growth,defend against phytopathogen infection and prevent plant disease occurrence (Caiet al.2015;Ashmitaet al.2018).The existing literature is mainly focused on the plant growth promoting rhizobacteria including the generaBacillusandPseudomonas(Otienoet al.2015;Gamezet al.2019).Some researchers have shown the plant growth promoting fungi such asAspergillus flavus,Penicillium chrysogenum,as well asTrichoderma koningiopsis,which also act as beneficial microorganisms in the promotion of plant growth and yield through systemic resistance induction and bioactive substance production (Adiyadolgoret al.2020;Saadet al.2020).According to previous studies,Trichodermaspp.may serve as prominent fungal species in promoting plant growth through increasing plant resistance and enhancing nutrient uptake,in addition to their role as biocontrol agents (Shuklaet al.2012;Pascaleet al.2017;Contreras-Cornejoet al.2018;Moyaet al.2020).

With the rapid development of high-throughput sequencing technology,an RNA seq-based approach is often applied to explore the mechanisms of plant growth promoting actions of microbes on host plants (Sugimura and Saito 2017;Liet al.2018).There are some reports on the transcriptional responses ofTrichoderma-inoculated host plants such as rice (Doniet al.2019),tomato (Coppolaet al.2019;De Palmaet al.2019) and cucumber (Yuanet al.2019).TheTrichodermatreatment usually leads to upregulation of a number of functional genes related to plant growth,stress tolerance and disease resistance at the transcriptome level.However,previous studies were mainly focused on the use ofTrichodermaspore suspensions.For further registration and application of the beneficial fungus as a microbial inoculation agent,a plant growth promotingTrichodermaisolate designated as TM2-4 was screened and prepared as a microbial agent after solid state fermentation.The present study was conducted to (i) characterize the species taxonomic identity of theTrichodermaisolate TM2-4 based on morphological characters and molecular methods;(ii)detect the beneficial biological activities,growth promotion effect,and rhizosphere colonization ability of the isolate TM2-4 on tomato plants;(iii) understand the gene expression pattern of tomato roots in response toTrichodermamicrobial agent inoculation at the transcriptional level.

2.Materials and methods

2.1.Isolation and screening of beneficial Trichoderma isolates

Rhizosphere soil samples were collected from healthy tomato plants in the Beijing suburb of Shunyi District in Chinaviathe diagonal method.Trichodermaisolates were obtained using the soil dilution plate technique and purified by the single spore method onTrichodermaselective medium(TSM) (Elad and Chet 1983).Biological characters including indoleacetic acid production,siderophore production,phosphate solubilization,and 1-aminocyclopropane-1-carboxylate (ACC) deaminase production,as well as seed germination promotion were assessed to screenTrichodermaisolates with plant growth promoting activities.

For the quantitative determination of indoleacetic acid(IAA) like substances,the obtainedTrichodermaisolates were cultured in Czapek liquid media supplemented with 500 mg L–1of L-tryptophan for 5 days at 150 r min–1and 25°C.The IAA concentration in the supernatant was calibrated based on a standard curve after 1 mL of the culture supernatant was added to 4 mL of Salkowski reagent(Gordon and Weber 1951).Trichodermaisolates were also incubated in Chrome azurol S (CAS) liquid media for the detection of siderophore production according to previous literature (Machuca and Milagres 2003).Quantitative calculation of phosphate solubilization ability was performed by growing theTrichodermaisolates in NBRIP liquid media at 25°C and 150 r min–1for 5 days (Nautiyal 1999;Achalet al.2007).ACC deaminase activity of theTrichodermaisolates was evaluated quantitatively by measuring the amount of a-ketobutyrate produced by the deamination of ACC (Viterboet al.2010).The cell-free culture filtrate ofTrichodermaisolate and its 10-,100-,200-,500-fold dilutions with sterile water were prepared (Zhaoet al.2018) for seed germination testing on tomatoviathe standard procedure of the International Seed Testing Association (ISTA 2005).

2.2.Morphological and molecular identification of the Trichoderma isolate

Morphological identification of theTrichodermaisolate was completed based on colony and microscopic characteristic observations according to the ISTH(International Subcommission on Trichoderma and Hypocera Taxonomy) after 5 days incubation at 25°C on potato dextrose agar (PDA) media (Gams and Bisset 2002).Molecular identification was conducted by multiple sequence analyses of three different phylogenetic markers,including the internal transcribed spacer (ITS) region (Whiteet al.1990),translation elongation factor 1-alpha (tef1-α) gene(Samuelset al.2002) and RNA polymerase II second largest subunit (rpb2) gene (Sawantet al.2017).The sequences of the rDNA-ITS,tef1-αandrpb2genes of theTrichodermaisolate were deposited in the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/).Phylogenetic analysis of the combined sequences of the rDNA-ITS,tef1-αandrpb2genes was computed using the maximum likelihood (ML)method in PAUP* version 4.0b10 (Swofford 2002).

2.3.Microbial agent preparation of the Trichoderma isolate by solid state fermentation

The obtainedTrichodermaisolate with biological activities was used to prepare the growth promoting microbial agent according to the revised method of Mauryaet al.(2012).Conidia suspension of theTrichodermaisolate (2.0×106CFU mL–1) was inoculated in a 250 mL Erlenmeyer flask containing 50 mL potato dextrose (PD) medium,and incubated at 28°C on a rotary shaker at 150 r min–1for 3 days in the dark.Afterwards,8 mL of theTrichodermaculture was transferred to a 500-mL beaker containing 100 g of solid fermentation medium (wheat bran,70 g;corn cob powder,30 g;mineral salt solution,60 mL).The beakers were covered with six layers of sterile gauze and incubated at 28°C for 7 days under 60% RH condition.Then the fermentation product was dried by natural wind,pulverized and screened through 80-mesh screens for further use asTrichodermainoculation microbial agent.

2.4.Growth promotion experiment of Trichoderma microbial agent on tomato plants

The solid fermentation microbial agent ofTrichodermaisolate(108cfu g–1conidia content) was used for the evaluation of its colonization ability and growth promotion effect on tomato plants (cv.Jiafen 16) in pots in a greenhouse.The experiment was conducted using a completely randomized design,with ten plants per treatment and three replications.Twenty-day-old plug seedlings were transplanted to pots with diameter of 12 cm.Tomato plants transplanted in the pots containing soil withTrichodermaagent added at a proportion of 10 g kg–1of soil were used as theTrichodermaagent inoculation treatment,while plants potted in soil withoutTrichodermaagent addition were taken as controls.Fiftyday-old plants were then carefully uprooted and washed under tap water.The capacities ofTrichodermaisolate to colonize and persist in the rhizosphere soil or root system of tomato plants were evaluated using soil dilution plate and tissue culture methods on TSM medium (Zhaoet al.2012).The plant growth promoting effect was determined by measuring major growth parameters including plant height,fresh weight,dry weight,number of leaves per plant,diameter of leaf canopy,diameter of stem base,root activity and leaf chlorophyll content (Wuet al.2019).

2.5.Transcriptome analysis of tomato roots treated with Trichoderma microbial agent

To assess tomato plant responses to theTrichodermaagent inoculation at the transcriptome level,RNA-seq analysis was conducted on the tomato roots treated with or withoutTrichodermamicrobial agent in the growth promotion assay.

2.6.RNA extraction,library construction and Illumina sequencing

Root tissues from tomato plants treated with or withoutTrichodermamicrobial agent were harvested for RNA extraction using QIAGEN RNeasy Plant Mini Kit (QIAGEN Co.,Ltd.,Shanghai,China).After quality and purity checking,the RNA samples were used for mRNA-seq library construction by NEBNext Ultra RNA Library Prep Kit (NEB,USA).Libraries were then indexed,pooled and sequenced on an Illumina Hi SeqTM2000 (San Diego,CA,USA) platform with the pair-end method at Majorbio Bio-pharm Technology Co.,Ltd.(Shanghai,China).

2.7.Transcriptome de novo assembly and annotation

After discarding adaptor sequences,empty reads and lowquality sequences,the remaining high-quality reads were mapped to theSolanum lycopersicumreference genome(Solanum lycopersicum,SL2.50:http://plants.ensembl.org/Solanum_lycopersicum/Info/Index).The unigenes were obtained by reference-based assembly and then used for BLAST search and annotation in the Gene Ontology (GO),Kyoto Encyclopedia of Genes and Genomes (KEGG),Eukaryotic Ortholog Groups (KOG),non-redundant protein sequences (NR),Swiss-prot protein,and Pfam database.The RNA sequencing data were deposited in the NCBI Sequence Read Archive (SRA) database.

2.8.Function annotation and enrichment analysis of differentially expressed genes (DEGs)

The fragments per kilobase per million reads method(FPKM) was used to determine the unigene expression levels (Trapnellet al.2010).The GO (http://www.geneontology.org) database was adopted to predict the functions of DEGs in relation to biological processes (BP),cellular component (CC) and molecular functions (MF) by the GO seq R package (Younget al.2010).The KEGG(http://www.genome.jp/kegg/) database was employed to determine the possible signal transduction or metabolic pathways in which the genes could possibly be involved by the KOBAS Software (Maoet al.2005).

2.9.qRT-PCR analysis of gene expression levels

Quantitative real-time PCR (qRT-PCR) was carried out to verify the expression patterns of ten genes that exhibited significant differencesviaRNA-seq analysis.Elongation factor 1-alpha (EF1α) gene was used as an internal reference.The primers for the selected DEGs were designed using the Primer Premier 5.0 Software.RNA samples from tomato roots treated with or withoutTrichodermamicrobial agent were both extracted and purified as described above.The cDNA was synthesized from 1 μg of purified RNA samples using KR106 Fast Quant RT Kit (Tiangen Biotech Co.,Ltd.,China) following the manufacturer’s instructions.The qRT-PCR amplification was performed using CFX 96 Real-Time PCR Detection System (Bio-Rad,Hercules,CA,United States) in a 20 μL system containing 2 μL of cDNA,1 μL of each primer (10 μmol L–1),10 μL of 2× TransStart Top Green qPCR SuperMix (TransGen,Beijing,China),and 7 μL of RNase-free water.The qRT-PCR was initiated at 95°C for 3 min,followed by 40 cycles of 15 s at 95°C,30 s at 60°C,and 72°C for 30 s.Relative expression levels of target genes were calculated by the 2−ΔΔCTmethod (Livak and Schmittgen 2001).

2.10.Statistical analysis

Values were expressed as mean±standard deviation and evaluated by analysis of variance (ANOVA) using SPSS Statistics 17.0 Software (SPSS Inc.,Chicago,United States).The RNA-seq data were analyzed on the free online platform of Majorbio Cloud Platform (www.majorbio.com).

3.Results

3.1.Biological characters of the obtained Trichoderma isolate

A total of 14Trichodermaisolates were obtained from the rhizosphere soil samples of tomato plants in the Beijing suburb of Shunyi district of China.One of them was a novel isolate,designated as TM2-4,with apparent plant growth promoting capabilities.A pink color was observed after the culture filtrate of TM2-4 isolate was added to the Salkowski reagent,indicative of production of indoleacetic acid (IAA) like substances.An average of (6.45±0.72) µg mL–1of IAA was detected for the isolate TM2-4.The isolate TM2-4 also showed siderophore production (52.3±1.6)% in Chrome azurol S (CAS) liquid medium and solubilization of phosphate (32.4 μg mL–1) in NBRIP medium.The isolate TM2-4 displayed no detectable 1-Aminocyclopropane-1-carboxylate (ACC) deaminase production activity.Tomato seeds treated with 500-fold dilution of culture filtrate of isolate TM2-4 exhibited a germination rate of 93.3% compared to the control (80%).The hypocotyl length,radical length and vigor index were significantly increased compared to the control by 28.7,19.4 and 62.1%,respectively,under 100-fold dilution treatment.

3.2.Morphological and molecular identification of the Trichoderma isolate

The morphological observation showed that mycelia of the isolate TM2-4 grew rapidly on PDA medium,covering the entire plate on the 3th day of incubation.The aerial hyphae were denser as white floss,which finally formed an arachnoid and cottony colony with dark green conidia on the surface.Conidiophores of the isolate TM2-4 were treelike,and composed of a main axis with several secondary branches,which terminated in phialides.The phialides of conidiogenous cells were ampullaceous or lagenate in shape,(3.5–7.5)×(2.5–3.8) μm in size.Conidia were ovoid to spherical,1.3–2.8 μm in diameter.Based on morphological and microscopic characteristics of the fungal colonies on PDA plates (Fig.1),the isolate TM2-4 was confirmed as belonging to aTrichodermaspecies.

Fig.1 Culture and microscopic characteristics of isolate TM2-4 on potato dextrose agar (PDA) medium.A,colony on PDA medium.B,conidiophores,phialides and conidia under microscopy (40×;scale bars=10 μm).

PCR amplification yielded fragments of approximately 582,578 and 1087 bp,respectively,for the rDNA-ITS region,tef1-αandrpb2genes of the isolate TM2-4.According to BLASTn results on NCBI,the ITS region of TM2-4 isolate had 99% nucleotide identity with those ofT.harzianum(MG572200,KX632495) andT.afroharzianum(KP009262).Thetef1-αandrpb2genes of isolate TM2-4 showed 99%similarity with those ofT.afroharzianumstrain LESF010(KT279008) and strain TRS835 (KP009149),respectively.Phylogenetic analysis of the combined data of rDNA-ITS region,tef1-αandrpb2genes showed that the isolate TM2-4 clustered together with the reference strain LESF010(KT278854,KT279008 and KT278940) and strain TRS835(KP009351,KP008787 and KP009149) ofT.afroharzianumwith 100% bootstrap support (Fig.2).The sequence data of rDNA-ITS region,tef1-αandrpb2genes of isolate TM2-4 were deposited in NCBI with GenBank accession numbers of MK779176,MK779175 and MK779174,respectively.Combining the molecular results with morphological characters,the isolate TM2-4 was identified asTrichoderma afroharzianum.

3.3.Growth promotion effect of T.afroharzianum microbial agent on tomato plants

The microbial agent was obtained by solid state fermentation of the obtainedT.afroharzianumisolate TM2-4.Theisolate TM2-4 displayed competitive rhizosphere colonization ability,since aT.afroharzianumcolony was recovered from the rhizosphere soil of tomato plants even 30 days post inoculation,with mean colony forming unit(CFU) numbers remaining at 2.92×107CFU g–1soil.The isolate TM2-4 was also detected from the root tissue of tomato plants.The pot experiment indicated that theT.afroharzianumTM2-4 microbial agent treatment gave considerably higher values of plant height,dry weight,number of leaves per plant and root activity,which were increased by 36.1,32.3,56.3 and 26.2%,respectively,compared with the control (P<0.05).The fresh weight,diameter of stem base,diameter of leaf canopy and chlorophyll content were also increased by 22.8,14.8,17.3 and 11.0% upon TM2-4 treatment (Fig.3).

3.4.Unigene annotation and identification of DEGs

High-throughput sequencing was used for the systematic analysis of gene expression profiles in tomato root treated with or withoutT.afroharzianumTM2-4 microbial agent.Approximately 47.24 and 45.23 million raw reads were acquired from the control (Trichodermaagent free,TCK)and the treatment (Trichodermaagent inoculation,TTM)groups,respectively.Large proportions (98.74 and 98.66%) of clean reads (46.64 and 44.62 million for TCK and TTM,respectively) were retained for assembly and downstream analysis (Table 1).Sequencing data were submitted to the NCBI Sequence Reads Archive database with SRA accession number PRJNA561218.The clean reads were mapped to the tomato reference genome,with detailed mapping output summarized in Table 1.Overall,the mapping ratios of the control andTrichodermatreatment groups were 93.60 and 92.41% (average value of uniquely mapped),respectively,suggesting high levels of gene expression in both groups.A total of 31 631 unique sequences were annotated based on blastx alignment searches of six public databases including GO,KEGG,NR,KOG,Swiss-prot and Pfam (Appendix A).We analyzed the datasets derived from transcriptome of tomato roots treated with or withoutT.afroharzianumTM2-4microbial agent.Gene expression analysis showed that 984 genes were significantly differentially expressed,including 584 up-regulated genes and 400 down-regulated genes.The list of differentially expressed genes (DEGs,FDR<0.05),including fold changes and annotation details,is presented in Appendix B.The 984 DEGs were apparently enriched in 44 terms of the three main GO categories (Appendix C),and in 206 pathways of the KEGG database (Appendix D).

Fig.2 Maximum parsimonious tree obtained from the combined rDNA-ITS,tef1-α and rpb2 sequence data of isolate TM2-4.Trichoderma longibrachiatum strain S328 was used as outgroup.Bootstrap support values >70% from 1 000 replications are shown at the nodes.Trees were inferred using the heuristic search option with Tree–Bisection–Reconnection (TBR) branch swapping.The numbers in the parentheses represent the GenBank accession numbers of rDNA-ITS,tef1-α and rpb2 genes of the corresponding strains.

Fig.3 Growth promotion effect of Trichoderma afroharzianum TM2-4 microbial agent inoculation on tomato plants under greenhouse conditions.Biological traits and physiological parameters were surveyed in fifty-day-old tomato plants.TCK and TTM,tomato roots treated without and with T.afroharzianum TM2-4 microbial agent inoculation,respectively.Error bars represent the standard deviations of three replicates.* denotes significant difference (P<0.05) in Trichoderma microbial agent treated samples compared to the control.

Table 1 Summary of trimming and read mapping results of the sequences obtained from tomato roots treated without or with Trichoderma afroharzianum TM2-4 microbial agent inoculation

3.5.GO function and KEGG pathway enrichment analysis of the DEGs

In evaluating the DEGs in terms of their predicted functions,the top 30 significant GO enrichment terms are displayed in Appendix E.To further clarify the definite functions of the DEGs and based on GO enrichment analysis,584 upregulated genes and 400 down-regulated genes were also assigned to the three categories.In the biological process(BP) category,the functions such as“hydrogen peroxide metabolic process”,“detoxification”,and“secondary metabolic process”were detected for the significant upregulated genes (Fig.4-A);while“cellular response to phosphate starvation”,“transmembrane transport”,and“cellular response to starvation”were detected for the down-regulated genes (Fig.4-B).The up-regulated genes were also significantly enriched in the cellular component(CC) category,such as“extracellular region”,“intrinsic component of plasma membrane”and“integral component of plasma membrane”(Fig.4-A).For the molecular functions(MF),“peroxidase activity”,“oxidoreductase activity”,and“glutathione transferase activity”were moderately enriched for the up-regulated genes (Fig.4-A).

We selected the twenty most abundant pathway entries of the 584 up-regulated and 400 down-regulated DEGs to display in the scatter plot graph (Fig.5).For the up-regulated DEGs,“phenylpropanoid biosynthesis”and“glutathione metabolism”were the most enriched KEGG pathways (Fig.5-A),while“vitamin B6 metabolism”was significantly enriched for the down-regulated DEGs(Fig.5-B).Biochemical synthetic and metabolic pathways,in which different genes were involved,were also determined through KEGG pathway enrichment analysis.The results indicated that the gene products were obviously associated with specific pathways,including phenylpropanoid biosynthesis and glutathione metabolism.For example,4-coumarate-CoA ligase (EC:6.2.1.12) and trans-cinnamate A4-monooxygenase (EC:1.14.14.91) are involved in the early stages of phenylpropanoid biosynthesis.In addition,several higher expressed enzymes,namely cinnamoyl-CoA reductase (EC:1.2.1.44),cinnamyl-alcohol dehydrogenase(EC:1.1.1.195),Peroxidase (EC:1.11.1.7),are concerned with the biosynthesis and modification of phenylpropanoids(Appendix F).KEGG enrichment analysis of the glutathione(GSH) metabolism showed that up-regulation of the glutathione peroxidase GPX (EC:1.11.1.9),phospholipidhydroperoxide glutathione peroxidase GPX4 (EC:1.11.1.12)and glutathione reductase (EC:1.8.1.7) were involved in the conversion of GSH to GSH disulfide as well as the opposite reaction (Appendix G).

Fig.4 Most highly enriched GO functions related to biological process,cellular component,and molecular function among Trichoderma afroharzianum TM2-4 microbial agent up-regulated (A) and down-regulated (B) differentially expressed genes (DEGs).

Fig.5 The top 20 enriched KEGG pathways of the up-regulated (A) and down-regulated (B) differentially expressed genes (DEGs)after the Trichoderma afroharzianum TM2-4 microbial agent inoculation.The vertical axis indicates different pathways,while the horizontal axis indicates the enrichment factor.Red indicates highly expressed genes,blue indicates genes with low expression.

3.6.qRT-PCR validation of the gene expression levels of the DEGs

To confirm the reliability of the RNA-seq data,the relative expression levels of ten DEGs related to phenylpropanoid biosynthesis (Solyc03g116910.2,Solyc12g005790.1),glutathione metabolism (Solyc01g099590.2,Solyc09g011630.2),substance and energy metabolism (Solyc10g078550.1,Solyc06g006080.2,Solyc04g080010.2),transporter activity(Solyc10g084120.1),and plant hormone signal transduction(Solyc04g080660.2,Solyc10g079640.1) pathways were confirmed using qRT-PCR with specific primers (Appendix H).As shown in Fig.6,the qRT-PCR results were highly consistent with the RNA sequencing analysis,supporting the high quality of the RNA sequencing datasets.

4.Discussion

This study demonstrated the obvious growth promotion ability ofTrichoderma afroharzianumTM2-4 microbial agent on tomato plants,which was closely related to successful survival and colonization of the miroorganisms in the tomato rhizosphere.In addition,the isolate TM2-4 used forTrichodermaagent preparation and tomato plant inoculation was proven to produce a variety of aforementioned bioactive substances.These are consistent with previous studies which declared that plant growth promoting characters of microbials commonly include the production of diverse chemical substances such as phytohormones,siderophore,phosphate solubilizing enzyme and ACC deaminase(Rodriguezet al.2008;Waniet al.2015).

High-throughput sequencing methods have revolutionized genomic and transcriptomic research with advantages of low cost and ultra-high data output.The RNA-seq method allowed us to undertake rapid gene expression analysis in plants interacting with rhizosphere microorganisms,which is especially applicable for plants with available reference genome sequences (Varshneyet al.2009;Camilios-Netoet al.2014).Trichodermaspp.are recognized as beneficial fungi that can systemically alter and influence the expression profile of numerous plant functional genes(De Palmaet al.2019;Yuanet al.2019).In this study,the RNA-seq method was used to detect the gene expression levels of tomato roots treated byT.afroharzianumTM2-4 microbial agent inoculation at the transcriptome level.The results indicated that theTrichodermaagent inoculation has brought about up-regulation of numerous genes,which may involve different signal transduction and metabolic process of tomato plants.

Fig.6 Comparison of the RNA-seq and qRT-PCR results validate the relative expression levels of differentially expressed genes(DEGs).The graph shows the Log2(fold change) in expression of genes in Trichoderma afroharzianum TM2-4 microbial agent inoculated tomato roots compared to the control.Error bars represent the standard deviations of three replicates.

According to the information obtained from GO function and KEGG pathway enrichment analyses,the up-regulated differentially expressed genes (DEGs) were mainly related to auxin signaling transduction,plant-type cell wall organization,and photosynthesis pathways.Previous studies have verified the capacity ofTrichodermato release elicitors that may induce auxin signal transduction,such as salicylic acid (SA),jasmonic acid (JA) and ethylene (ET),which are often associated with induced systematic resistance within the plants (Nawrocka and Malolepsza 2013).The cell wall is also critical in the plant defense system considering its function of protecting plants from invasion of biotic or abiotic stress (Underwood 2012;Malinovskyet al.2014).The upregulation of these genes related to plant defense activity can be considered as an indication of better plant fitness and a greater ability of the plant to resist external influences(Doniet al.2019).In addition,the up-regulation of genes associated with chlorophyll biosynthesis and photosynthesis also demonstrated an increase of the tomato plants’ capacity to transport and accumulate photosynthetic products,which has significant roles in plant growth and development (Doniet al.2019).We also found that theT.afroharzianumTM2-4 treatment could induce the up-regulation of phenylpropanoid biosynthesis and glutathione metabolism related genes,which usually play an important role in reinforcing plant cell walls and improving plant defenses through modulation of the plant secondary metabolism (Benet al.2017).Hence,the growth promotion mechanism ofT.afroharzianumTM2-4 on tomato plants mainly manifests as growth and metabolism enhancement,immune defense optimization,and induced systematic resistance stimulation.

A decreased application rate of chemical fertilizers combined with biological fertilizers,such as microbial agents,can give the same levels of plant growth and yield as those obtained with the full use rate of chemical fertilizers(Caiet al.2015).Therefore,plant growth promoting fungi based microbial agents should be evaluated and used as components of integrated management strategies in plant cultivation.In this study,we demonstrated significant stimulation of plant growth and the corresponding mechanism at the transcriptomic level in tomato plants byT.afroharzianumTM2-4 microbial agent inoculation.The isolateTM2-4 was proven to have the potential of application as plant growth promoting fungi (PGPF) in tomato plantation.Further research will focus on theformulation development and optimization ofT.afroharzianummicrobial agent products,as well as the chemical fertilizer usage reduction effect in field application.

5.Conclusion

In the present study,we described the tomato plant growth promoting effect ofTrichoderma afroharzianumisolate TM2-4,a beneficial fungus previously obtained from tomato rhizosphere soil.RNA-seq technology revealed the transcriptome profiling of tomato plants byT.afroharzianumTM2-4 microbial agent inoculation.The results indicated that the growth promotion effect of isolate TM2-4 was associated with up-regulated expression of hundreds of genes which were implicated in nutrition and energy metabolism,antioxidant activity,auxin signaling pathway,and immune defense activity.The research provided an in-depth understanding of the mechanism of action of the rhizosphere competentT.afroharzianumisolate TM2-4 in relation to tomato plant growth promotion.

Acknowledgements

This research was supported by the Youth Research Fund of Beijing Academy of Agriculture and Forestry Sciences,China (QNJJ201814),the National Key R&D Program of China (2017YFD0201102),and the Beijing Key Laboratory of Green Control of Fruit Tree Diseases and Pests in the North China (BZ0432).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm