The disease resistance potential of Trichoderma asperellum T-Pa2 isolated from Phellodendron amurense rhizosphere soil

Zeyang Yu · Zhihua Liu,3 · Yuzhou Zhang · Zhiying Wang

Abstract To promote the application of Trichoderma, many countries have collected Trichoderma resources.In the present study, nine isolates were isolated from a rhizosphere soil of Phellodendron amurense and were identified as three species: Trichoderma brevicompactum (one isolate), T.asperellum (two isolates), T.atroviride (six isolates).Dual culture experiments showed that T.asperellum T-Pa2 grew fast and produced the best inhibition rates against six tested pathogens (80.25-91.65%) via competition and mycoparasitism.Populus davidiana × P.alba var.pyramidalis Louche (PdPap poplar) was treated with T-Pa2, which increased the catalase activity, nitrate reductase activity, and content of osmosis molecules significantly (P ＜ 0.05).Meanwhile, induction by T-Pa2 increased the resistance of PdPap poplar against Alternaria alternata via modulating the expression of salicylic acid, jasmonic acid, and auxin transduction pathway genes.The results will form the basis for the collection and application of biocontrol agents in forestry.

Keywords Biological control · Trichoderma identification · Antifungal property · Enzyme activity

Introduction

The important fungus,Trichodermaspp., has been used commercially worldwide, and is always found in the soil or rhizosphere of plants (Druzhinina and Kubicek 2013).As an effective biocontrol agent,Trichodermaplays a key role in forestry and agriculture through the inhibition of pathogens, promotion of plant growth, and induction of plant resistance (Yuan et al.2019).For example, the gene encoding the glucose transporter (Gtt1) inT.harzianumCECT 2413 is expressed under low nutrient conditions, which is important for competence with other pathogens (Benítez et al.2005).T.harzianumETS 323 inhibits the growth ofBotrytis cinereaand causes hyphal lysis (Cheng et al.2012).T.asperellumstrains Bt2, Bt3, and T25 could effectively inhibit the growth of sixVerticillium dahiliaestrains; however, the extracellular compounds of Bt3 and T25 showed rates of higher abiosis, suggesting that inhibition mechanisms of the three strains are different (Carrero-Carrón et al.2016).In addition,Trichodermaspecies also have beneficial effects on plants because of their ability to enhance plant’s immunity against different pathogens.T.asperellumBt3 and T25 increased the resistance of olive plants toV.dahiliae, significantly decreasing the severity of symptoms (Carrero-Carrón et al.2016).T.atrovirideenhanced the resistance ofPopulus: The leaves inoculated with the pathogens showed smaller specks (Raghavendra and Newcombe 2013).

For many years,Trichodermahas been packed into commercial products as biopesticides, biofertilizers, and growth promoters because of its multifaceted mechanisms (Woo et al.2014); however, the effect ofTrichodermais influenced by complicated environments or species differences.Even different strains from the same species have different biocontrol abilities; therefore, it is important to collect a variety ofTrichodermaresources to find more activeTrichodermastrains, a job that has been performed in many countries for many years (Zeng et al.2016; Swain et al.2018).For example, 183 isolates were obtained from Mexico, Guatemala, Panama, Ecuador, Peru, Brazil, and Colombia, and identified as 29 species (Hoyos-Carvajal et al.2009).In the Mirzapur district of Uttar Pradesh, 27Trichodermastrains were isolated from rhizosphere of different plants (Tripathi et al.2015).

Trichodermahave also been collected in China for many years; however, most of these studies have focused on south or southwest China (Sun et al.2012).Heilongjiang Province, located in the northeast of China, is a key area of forestry and agriculture, where the use of pesticides and chemical fertilizers has become common and normal.However, the collection of the biocontrol agentTrichoderma, especially in forestry areas, is limited.Poplar species are important for wood production, and are planted worldwide.Poplar is also a model organism to study woody plants (Guo et al.2018).

In the present study, we aimed to isolate robustTrichodermastrains fromPhellodendron amurenserhizosphere soil samples in Heilongjiang Province in China.The antagonistic potential and the beneficial effects on poplars of the isolatedTrichodermaspp.were measured.The present study provided a good method to collectTrichodermaresources, and preliminarily investigated the interaction ofTrichodermaisolates with pathogens and plants.

Materials and methods

Isolation and identification of Trichoderma

Trichodermafungi were isolated from 13 soil samples ofP.amurenserhizosphere soil (126.63° E, 45.72° N) in Harbin, Heilongjiang Province, China.The soil samples were collected in November 2018.For each soil sample, a 5-g subsample were diluted to concentrations of 1 × 10-1, 1 × 10-2, 1 × 10-3, and 1 × 10-4using sterile water.Each dilution was coated on three Rose Bengal medium plates and cultured at 28 °C for 1-4 days.During this time, the plates were observed every 24 h, and the colonies ofTrichodermawere transferred to Potato Dextrose Agar (PDA) medium and incubated at 28 °C.When the isolate had been incubated for 48-72 h, the white mycelium was transferred to glass slide, stained using a blue dye solution (biohao Biotechnology Co., Ltd., Beijing, China, Catalog No.C0710), and observed under a microscope (Leica DM750, Wetzlar, Germany).When the isolate had been cultured for 7 d, images of the colonies were obtained.Phenotypic identification of theTrichodermawas based on the microscopic characteristics and a comparison of the colony images comparison, as described by a previous study (Gams and Bissett 1998).

For each species identified through morphological examination, one isolate was chosen for molecular identification to verify the result.The isolatedTrichodermawas shaking cultured for 48 h and the hyphae were collected onto filter paper, washed with distilled water, and the mycelia were stored at-80 °C.The DNA was extracted using a fungal DNA extraction kit (OMEGA Biotek, Beijing, China).Using this DNA as a template, the internal transcribed spacer (ITS) region was amplified using primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′; Rogers and Bendich 1994), while the translation elongation factor 1-α (tef1-α) region was amplified using primers EF1-728F (5′-CAT CGA GAA GTT CGA GAA GG-3′) and EF1-986F (5′-TAC TTG AAG GAA CCC TTA CC-3′; Druzhinina et al.2005).PCR amplification of ITS andtef1-αsequences was performed using a 50 μL PCR reaction comprising 10 × PCR buffer, 5 μL; 2 μmol L-1dNTPs, 1 μL; Taq DNA polymerase, 0.5 μL; 20 μmol L-1primer one, 1 μL; 20 μmol L-1primer two, 1 μL; 100 ng μL-1DNA template, 0.5 μL; and ddH2O, 41 μL.The PCR procedure was: 94 °C for 5 min; followed by 35 cycles of 94 °C for 1 min, 50 °C for 30 s, and 72 °C for 1 min; and 72 °C for 7 min.The purified ITS andtef1-αPCR products were obtained using a Gel Extraction Kit (Promega, Madison, WI, USA, Kit No.A9281) and subjected to directs automated sequencing using fluorescent terminators on an ABI 377 Prism Sequencer (Sangon Biotech, Shanghai, China).

The sequences were analyzed using the CLUSTAL W program (Thompson et al.1994), bioinformatic tools of International Subcommission ofTrichodermaandHypocrea(ISTH, www.isth.info), and the BLASTN algorithm from the National Centre for Biotechnology Information (NCBI).A phylogenetic tree was constructed using the Neighborjoining (NJ) method with 1000 bootstrap replications in the MEGA 7.0 package (Kumar et al.2016).

Antagonism of Trichoderma against six phytopathogens

TheTrichodermaisolate from each species with the fastest growth on PDA media was chosen for antagonism analysis.The tested phytopathogens wereF.camptocerus,F.solani,A.alternata,F.oxysporum,G.applanatum, andB.cinerea, which were stored at the Key Laboratory (Forest Pest and Disease) of the State Forestry Administration in the Northeast Forestry University.Spores suspensions of the nineTrichodermaisolates were prepared in sterile water at 1 × 106spores mL-1.Then, 3 μL of the spore suspensions were inoculated on the edge of PDA in petri dishes, and the radius of colony after cultured at 28 °C for 48 h was recorded.The experiment performed three times independently.The antagonism betweenTrichodermaand the phytopathogens was tested using dual culture method on PDA media.A 5-mm diameter mycelial disc of antagonistic fungi from the margin of the colony was placed on the edge of the plate, and a mycelial disc of the phytopathogen was placed at the opposite side of the plate at an equal distance.Completely randomized three petri dishes were used for each antagonist as the experimental design.After 12 days of incubation, the level of antagonism was evaluated using a modified version of a previously published method (Popiel et al.2008): If theTrichodermacould fully overgrow on the pathogen, the score was 8; if theTrichodermaoccupied 85% of the plate’s surface area, the score was 6; if theTrichodermaoccupied 70% of the plate’s surface area, the score was 4; if theTrichodermaonly occupied 50% of the plate’s surface area, the score was 0.The interaction zone of the pathogens withTrichodermathat obtained the highest score was further observed under a microscope and photographed.

Plant resistance induction by Trichoderma

Based on the evaluation above, the most potent isolate (T.asperellumT-Pa2) was chosen and its biocontrol effect was further tested in a greenhouse experiment.Populus davidiana×P.albavar.pyramidalisLouche (PdPap poplar) seedlings were cultured aseptically (Guo et al.2018) for 30 days and then transferred to pots.The seedlings were cultured in soil for 30 days under a 16-h light/ 8-h dark cycle at 25 °C, with a relative humidity of 65%.Plants of similar height were chosen for analysis.

Firstly, the stress-related indicators were measured.The treatment group (marked asPopulus-T) was watered and sprayed with a spore suspension ofT.asperellumT-Pa2 (107conidia mL-1), and the control group (marked asPopulus-C) was watered and sprayed with water lacking conidia.After treatment 48-72 h, the leaves of PdPap poplar were harvested and used to measure catalase (CAT) activity, nitrate reductase (NR) activity, and the contents of soluble sugars and soluble proteins.CAT activity was measured by testing the decrease of absorption at 240 nm, and calculated using the extinction coefficient (40 mM-1cm-1) for H2O2(Guo et al.2010).Soluble sugar was measured using the anthrone method, with sucrose used as the standard (Guo et al.2010).NR activity was measured according to the method described by Jaworski (1971).Soluble protein was measured using the Coomassie brilliant blue G-250 staining method and standard curve was constructed using bovine serum albumin (Jones et al.1989).

Secondly, the disease resistance improvement of PdPap poplar caused byTrichodermawas evaluated.The seedlings were divided into three groups and marked as Group-T, Group-T + A, and Group-A.The seedlings in Group-T and Group-T + A were treated with 300 mL of a spore suspension of candidateTrichoderma, while the seedlings in Group-A were treated with 300 mL water.After 3 days, the seedlings of the three groups were punctured, and those in Group-T + A and Group-A were inoculated withA.alternata.Plant morbidity was observed after 12 days.

Differential expression of hormone responsive genes induced by candidate Trichoderma and A.alternata

To test the changes in the expression of hormone genes caused by candidateTrichodermaT-Pa2, the seedlings in Group-T and Group-T + A were treated with 300 mL of a spore suspension of T-Pa2, while the seedlings in Group-A were treated with 300 mL water.After treatment for 0, 7, and 28 h, the seedlings in Group-T were harvested and stored at-70 °C.After treatment by T-Pa2 for 3-4 days, the seedlings in Group-T + A and Group-A were inoculated withA.alternata, harvested at 0, 7, and 28 h after inoculation, and stored at-70 °C.Total RNA was extracted using the CTAB method (Ji et al.2016) and reverse transcribed to cDNA using an RNA Kit (Bio-Take RP3301, Beijing, China) and a reverse transcription kit (Takara RR047A, Dalian, China).The 20 μL PCR system for quantitative real-time reverse transcription PCR (RT-qPCR) comprised 2 × SYBR Green Realtime, 10 μL; 10 μM primer, 0.5 μL; cDNA, 2 μL; and RNAse Free ddH2O, 7 μL.The PCR procedure was 94 °C for 30 s; 35 cycles of 94 °C for 2 s, 59 °C for 30 s and 72 °C for 5 min.The transcription level was calculated using 2-ΔΔCtmethod (Livak and Schmittgen 2001).Actin,HAF01, andTublinwere chosen as reference genes.The candidate hormone responsive genes (including salicylic acid pathway geneNPR1, jasmonic acid pathway genesJAR1andMYC2, and auxin pathway genesIAA10andGH3.17) were chosen according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https:// www.kegg.jp/ dbget-bin/ www_ bget? map04 075).The primers were designed using primer premier 6.0 (Premier Biosoft, Palo Alto, CA, USA) and are shown in Table 1.

Table 1 Primers used for RT-qPCR

All experimental data were subjected to analysis using SPSS software version 19.0 (IBM Corp., Armonk, NY, USA).Statistical significance was determined using an independentsample T test (P＜ 0.05).

Results

Isolation and identification

Nine isolates from ten soil samples were isolated and identified (Fig.1).With reference to a previous study (Gams and Bissett 1998), these isolates were preliminary identified asT.brevicompactum(one isolate),T.asperellum(two isolates), andT.atroviride(six isolates).

Fig.1 Morphological identification of Trichoderma.Trichoderma strains were cultivated on PDA medium, and the hyphae were analyzed under a Leica DM750 microscope at 48 h after inoculation.The macro morphology was analyzed at 7 d after inoculation.a1: Macro morphology of T.brevicompactum. b1: Macro morphology of T.asperellum.c1: Macro morphology of T.atroviride.a2: Micro morphology of T.brevicompactum.b2: Micro morphology of T.asperellum.c2: Micro morphology of T.atroviride.The ruler in a1, b1 and c1 represents 3 cm; the ruler in a2, b2 and c2 represents 50 μm

Statistical analysis

The morphological identification was supplemented by molecular identification based on ITS andtef1-αsequence analysis.The ITS sequences were submitted to the ISTH database, and the alignments confirmed the morphological identification, the sequences of the isolates matched the standard sequences ofT.brevicompactum,T.asperellumandT.atrovirideseparately.Furthermore, BLAST analysis showed that the ITS sequences ofT.brevicompactumhuang1, T.asperellum-huang2 andT.atroviride-huang3 were successfully amplified, and showed 94-100%, 97-100%, and 97-100% identity, respectively, to the available sequences in the NCBI public database.Thetef1-αsequences ofT.brevicompactum-huang1 andT.asperellum-huang2 were amplified successfully, and showed 93.66-98.87% and 98.75-99.38% identity, respectively, to the available sequences in the NCBI public database.These sequences were submitted to GenBank, and the accession number of the ITS sequences were MK377320 (T.brevicompactum-huang1), MK377321 (T.asperellumhuang2), and MK377322 (T.atroviride-huang3), while the accession numbers of thetef1-αsequences were MW009739 (T.brevicompactum-huang1) and MW009738 (T.asperellum-huang2).

A phylogenetic tree was constructed using 22 ITS sequences including the three isolates (T.brevicompactumhuang1, T.asperellum-huang2 andT.atroviride-huang3) and the sequences of 19 otherTrichodermaobtained from the NCBI or ISTH databases (Fig.2a).Another phylogenetic tree was constructed using 24tef1-αsequences, including the two isolates (T.brevicompactum-huang1 andT.asperellum-huang2) and the sequences of 22 otherTrichodermaobtained from the NCBI or ISTH databases (Fig.2b).The sequences in same clade are considered to have a close genetic relationship, and the results agreed with the morphological identification and the alignments obtained at ISTH.

Fig.2 Phylogenetic tree constructed based on ITS and tef1-α sequences of Trichoderma strains.a The 22 ITS sequences, including three isolates from the present study and other 19 closely-related Trichoderma species.b The 24 tef1-α sequences, including two isolates from the present study and other 22 closely-related Trichoderma species.The sequences of closely-related Trichoderma species were obtained from the NCBI database (https:// blast.ncbi.nlm.nih.gov/) or the ISTH (http://www.isth.info/) database, the accession numbers of the sequences are provided in brackets

Antagonism assay

According to the radius of the colony on PDA medium (Fig.3), T-Pa2 was the isolate ofT.asperellumwith the highest growth rate, while T-Pa5 was the isolate ofT.atroviridewith the highest growth rate.T-Pa1 was the only isolate inT.brevicompactum; therefore, accompanied by T-Pa2 and T-Pa5, these three isolates were chosen for the antagonism assay.

Fig.3 Growth rate of Trichoderma isolates cultivated on PDA plates.Data are the radii of colonies at 48 h and are presented as mean ± standard deviation from three independent experiments

The antagonism assay showed that isolates T-Pa1, T-Pa2, and T-Pa5 have varying degrees of antagonistic responses againstF.camptocerus,F.solani,A.alternata,F.oxysporum,G.applanatum, andB.cinerea(Fig.4a).Growth of pathogens were obviously inhibited only after contact with hyphae ofTrichoderma.After 12 days of incubation, the inhibition rate of T-Pa1 againstF.camptocerus,F.solani,A.alternata,F.oxysporum,G.applanatum, andB.cinereawere 80.25%, 80.25%, 84%, 85.73%, 87.36% and 88.89%; those of T-Pa2 were 80.25%, 87.36%, 90.32%, 82.17%, 80.25%, and 91.65%; while the inhibition rates of T-Pa5 were 80.25%, 55.56%, 80.25%, 55.56%,76.1%, and 84%, respectively.The qualitative evaluation showed that T-Pa2 was the best antagonist (Fig.4b), it captured the space fast, and could overgrew completely on the five of six pathogens with mycoparasitism.The interaction zones between T-Pa2 and the five tested pathogens were examined microscopically, except forF.camptocerus, and coiling and hook-like structures were observed at the interaction zone (Fig.4c).

Fig.4 Pathogen inhibition experiment using three Trichoderma isolates against six pathogen strains.Mycelia disc of Trichoderma and the pathogens were placed at opposite sides of PDA plates and incubated at 28 °C.A Antagonism assay of Trichoderma (left) and phytopathogens (right) (12 d).T-Pa1: T.brevicompactum, T-Pa2: T.asperellum, T-Pa5: T.atroviride; a: F.camptocerus, b: F.oxysporum, c: A.alternata, d: F.solani, e: G.applanatum; f: B.cinerea.B Qualitative evaluation of antagonism (12 d): Trichoderma can fully overgrow on the pathogen, giving a score of 8; Trichoderma occupied 85% of the plate’s surface area, giving a score of 6; Trichoderma occupied 70% of the plate’s surface area, giving a score of 4; Trichoderma only occupied 50% of the plate’s surface area, giving a score of 0.c Micrographs of the interaction zone between T.asperellum T-Pa2 and five pathogens.a: F.oxysporum, b: A.alternata, c: F.solani, d: G.applanatum; e: B.cinerea.The ruler represent 200 μm.An arrow points at the coiling and hook-like structure

Plant resistance induction

After treatment byT.asperellumT-Pa2, the CAT activity, NR activity, and soluble sugar and soluble protein contentswere detected (Table 2).The CAT activity can reflect the plant’s ability to adapt the stress, with a higher CAT activity indicating a stronger ability to eliminate reactive oxygen species (ROS) and further adapt to stress (Chen et al.2019).The PdPap poplar seedlings in thePopulus-T group had a higher CAT activity than those in thePopulus-C group (P＜ 0.05).NR activity is one of the indexes that reflect the rate of nitrogen assimilation, with higher NR activity indicating higher metabolism and better growth conditions (Chen et al.2019).We found that the seedlings in thePopulus-T group had significantly higher NR activity than those in thePopulus-C group (P＜ 0.05).Soluble sugars and soluble proteins are key players in the regulation of osmotic balance and protect the integrity of membranes, such that a higher content of soluble sugars and soluble proteins indicates a stronger immune system (Abd-Allah et al.2015; Hoekstra et al.2001).We found the seedlings in thePopulus-T group had higher contents of soluble sugars and soluble proteins than those in thePopulus-C group (P＜ 0.05).These results indicated that T-Pa2 increased the stress adaptation of PdPap poplars.

Table 2 Physiological index of leaves of PdPap poplars

At 12 d after inoculation ofA.alternata, the seedlings in Group-T (inoculation of water, served as control) showed good growth without any disease spots, while the disease spots appeared on the leaves of poplars in Group-T + A and Group-A.However, although there were diseased spots on the leaves of the poplar in Group-T + A, the disease spots did not continue to grow after their appearance.Compared with the poplars in Group-A, the diseased spots in Group-T + A were smaller (Fig.5).These results demonstrated that T-Pa2 could successfully induce resistance of PdPap poplar toA.alternataand should be further investigated.

Fig.5 The morbidity of PdPap after inoculation with Trichoderma asperellum T-Pa2 and Alternaria alternata for 12 days.a Group-T: the poplars only treated with T-Pa2 for 3 d, after which the leaves were punctured and treated with water for 12 d; b Group-A: poplars treated with water for 3 d, after which the leaves were punctured and inoculated with A.alternata for 12 d; c Group-T + A: poplars treated with T-Pa2 for 3 d, after which the leaves were punctured and inoculated with A.alternata for 12 d; d Morbidity of the leaves

Di fferential expression of hormone-responsive genes

The transcription level of five hormone-responsive genes (NPR1,JAR1,MYC2,IAA10, andGH3.17) were tested using RT-qPCR (Fig.6).In the salicylic acid (SA) pathway, theNPR1gene is encoding a pathogenesis-related factor that can regulate the expression of downstream resistance genes and realize the establishment of systemic resistance in plants.The expressions ofNPR1increased over time in the three groups; however, the transcript level ofNPR1in the Group-T + A was upregulated faster and to a higher level compared with those in the other two groups.

Fig.6 The transcription levels of five hormone responsive genes in PdPap seedlings.T: The seedlings in Group-T were treated with 300 mL of a spore suspension of Trichoderma asperellum T-Pa2; a: The seedlings in Group-A were treated with 300 mL of water, and after 3-4 d, they were inoculated with Alternaria alternata.T + A: the seedlings in Group-T + A were treated with 300 mL of a spore suspension of T-Pa2, and after 3-4 d, they were inoculated with A.alternata.a Salicylic acid pathway gene NPR1; b Jasmonic acid pathway gene JAR1; c Jasmonic acid pathway gene MYC2; d Auxin pathway gene IAA10; e Auxin pathway gene GH3.17

Jasmonic acid (JA) is an important signaling molecule in the induced defense response of plants; therefore, the transcript levels of theJAR1andMYC2genes in the JA pathway were tested.The expression ofJAR1andMYC2showed similar trends in Group-T + A and Group-A.In Group-T + A, the expression levels ofJAR1andMYC2increased rapidly at 7 h and were continuously upregulated upto 28 h, showing expression level increases of 23.49and 24.93times, respectively.While in Group-A,JAR1andMYC2both showed a trend of an initial slight downregulation, followed by upregulation.Their peak transcript levels both appeared at 28 h, increasing by 22.83and 23.33times, respectively.In Group-T, the expression of the two genes differed.The expression ofJAR1gene was slightly downregulated at 7 h and 28 h, and the change was not significant; however, theMYC2gene was markedly and increasingly upregulated.

Auxin pathway genes play an important role in the regulation of plant growth.In this study, two auxin genes,IAA10andGH3.17, were studied.The expression ofIAA10showed negative regulation in Group-T + A; however, in Group-A and Group-T, it showed a trend of initial negative regulation, followed by positive regulation.The transcript level ofGH3.17showed a trend of initial negative regulation and then upregulation in the three groups; however, the upregulation ofGH3.17in Group-T + A was the strongest, increasing by 20.95times at 28 h.

The results showed that induction by T-Pa2 altered the expression of hormone-responsive genes.In addition, the PdPap poplars acquired systemic resistance and showed a sensitive and strong response to pathogens.

Discussion

Currently, many countries, such the United States, Canada, and Australia have collectedTrichodermaresources.Chinese researchers have also isolatedTrichodermain China since the 1930s; however, they were mostly from middle and low latitudes area (Sun et al.2012), China is a vast country, and the geomorphology and climate can vary between two region, thus the effects ofTrichodermaare always limited by geographic and climatic conditions (Gajera et al.2016).Therefore, continuous collection ofTrichodermafrom different regions might solve the problem of geography-based efficiency loss effectively.In this study, nineTrichodermawere collected fromP.amurenseforest in Heilongjiang Province, one isT.brevicompactum, two areT.asperellumand six were identified asT.atroviride.T.atroviridewas the dominant species, which was similar to the results of previous research (Zhou et al.2019), in whichT.atroviridewas the dominant isolate fromJuglans mandshurica,Acer saccharum,Populus simonii, andUlmus pumilaforests in Heilongjiang Province.While the number ofT.brevicompactumisolates was the fewest (one isolate), which was consistent with the isolation results for lilac, Manchurian ash, and banana (Liu et al.2020; Zhou et al.2019; Xia et al.2011).Therefore, we speculated that the root exudates and rhizosphere environment of different plants are different, andTrichodermahas different preferences for such environments, which ultimately determines the distribution and abundance ofTrichoderma.Identification ofTrichodermaincludes both morphological and molecular methods (John et al.2015).In the present study, molecular identification using two DNA barcodes (ITS sequences andtef1-αsequences) was combined with detailed morphological identification for verification.

A number of species ofTrichodermae.g.,T.harzianum,T.viride, andT.atroviridehave been reported to act as biocontrol agents against plant pathogens (Kumar et al.2012).One of the biocontrol mechanisms is competing and capture the growth site (Contreras-Cornejo et al.2016), and the ability to grow fast is the dominant advantage for competing for space and nutrients (Ben Amira et al.2017).Therefore, growth rate was a dominant factor to filter the biocontrolTrichodermain the present study.Consequently, we chose the isolate with the highest growth rate to assess potential antagonism.In previous research, the inhibition byT.asperellum(Tr10) againstSclerotinia rolfsiishowed an inhibition rate of 79.63% (John et al.2015).T.asperellumTWD1 was also reported to inhibit the growth ofSclerotinia rolfsiisignificantly (inhibition rate: 69.6%); however, it could not inhibit the growth ofColletotrichum gloeosporioidesandC.capsiciefficiently, with inhibition rates of 40% and 38.9%, respectively (Kumar et al.2012).In this study, T-Pa2 (T.asperellum) exhibited a strong and varied control effect on six different pathogens (inhibition rates: 80.25-91.65%), and overgrow the mycelia of five pathogens through surround coiling and hook-like structures.T-Pa5 (T.atroviride) could only overgrowA.alternataandB.cinerea, while an isolateT.atroviridewas reported to overgrowF.graminearumand was further considered as a potential antagonistic fungus (Sch?neberg et al.2015).T.brevicompactumwas reported to produce trichodermin, which had a good inhibition effect onB.cinereaandR.solani; the effective concentrations of trichodermin causing a 50% inhibition of pathogen growth were 2.02 μg mL-1and 0.25 μg mL-1, respectively (Shentu et al.2014).In this study, T-Pa1 (T.brevicompactum) could efficiently inhibit the growth of six pathogens, with inhibition rates ranging from 80.25 to 88.89%; however, T-Pa1 could not mycoparasitize any of the tested pathogens, thus competition and antibiosis might be the major method by which T-Pa1 inhibits the growth of other fungi.Compared with T-Pa2, the biocontrol abilities of T-Pa1 and T-Pa5 were limited.

Aerobic metabolism will produce ROS, which is triggered by stress conditions.Excess ROS will damage the cell; however, it can be eliminated by enzymes such as ascorbate peroxidase (APX), CAT, and polyphenol oxidase (PPO) (Chen et al.2019; Foyer 2018).After induced byT.pseudoharzianumT17, the CAT activity of PdPap poplar was increased 14-fold, which decreased the harm of ROS (Zhou et al.2019).The polyphenol oxidase of tomato treated byT.asperellum(MSST) was increased, and the plant defense againstF.oxysporumwas enhanced, the reduction in the incidence of the plant up to 85% (Patel and Saraf 2017).In the present study, the CAT activity of PdPap poplar was also significantly increased (P＜ 0.05) after treatment with T-Pa2, which suggested successful induction of resistance of PdPap poplar.Reactive nitrogen species (RNS) are produced when plants interact with pathogens (Cánovas et al.2016).InoculatingPhytophthora infestanson potato caused rapid accumulation of RNS (Abramowski et al.2015).In addition,ArabidopsisshowedF.oxysporum-induced RNS accumulation (Gupta et al.2014).RNS signaling is an important positive factor in the interaction between plants and pathogens, which is mainly mediated by NR and nitric oxide synthases (Chen et al.2019; Lindermayr 2017).The NR activity of cucumber treated byF.oxysporumonly increased by 1.5 fold, and inoculation withT.harzianumameliorated this increase, which caused inhibition of NO accumulation; however, the NR activity of cucumber treated withT.harzianumonly did not change, while the levels of other enzymes sharing similar functions with NR decreased (Chen et al.2019).However, in our study, the NR activity of PdPap poplar treated withT.asperellumT-Pa2 increased significantly (P＜ 0.05), which indicated that RNS signaling was induced.This was different for previous research and might be caused by plant species and strain differences.In higher plants, osmotic molecules, such as proline, soluble sugars, and soluble proteins, are important indicators of stress tolerance (Abd-Allah et al.2015; Hoekstra et al.2001).The contents of soluble protein and proline in tomato increased significantly, by 32.08% and 26.7%, respectively, after induction byT.harzianum, which enhanced the stress tolerance of tomato (Alwahibi et al.2017).When faba bean was under saline stress, the soluble protein content increased with the increase in salinity; however, treatment withT.harzianumdecreased this accumulation of soluble proteins.These suggestedTrichodermastrains have different effects on the soluble protein content under different conditions.In our study, the plants treated with the isolate T-Pa2 (T.asperellum) had significantly higher contents of soluble sugars (P＜ 0.05) and soluble protein than those in the control (P＜ 0.05), which suggested that T-Pa2 increase plant stress tolerance by increasing the contents of soluble sugars and soluble proteins.

Plant hormones are important in the defense against pathogens; therefore, the transcription levels of hormone genes in PdPap were investigated.In Oogata-Fukuju tomato, treatment withT.virenssignificantly increased the expression of JA pathway genePDF1(by 6.05-fold,P＜ 0.001) and SA pathway genePR1a(by 10.10-fold,P＜ 0.01), and this treatment also enhanced the resistance of tomato toFusarium(Jogaiah et al.2017).When olive trees were treated withT.harzianumThs97, the inoculation ofF.solanitriggered the expression of genes in the JA and SA pathways; however, the separate inoculation could not modulate the expression of the genes significantly (Ben Amira et al.2017).In our study, separate inoculation of T-Pa2 (Group-T) andA.alternata(Group-A) caused upregulated expression ofNPR1in the SA pathway andMYC2in the JA pathway.The combined inoculation (Group-T + A) triggered stronger upregulation of these two genes.We found that the JA pathway geneJAR1was not modulated significantly by T-Pa2 (Group-T); however, its expression was upregulated signifciantly in Group-A and Group-T + A, and the expression modulation in Group-T + A was faster and stronger.The result was similar to that in previous research, which reported that co-inoculation withT.harzianumThs97 andF.solaniFso14 further enhanced the plant response compared with single inoculation (Ben Amira et al.2017).Combined with the morbidity results, these results indicated that after inoculation with T-Pa2, the resistance of PdPap poplar againstA.alternataincreased via the activation of JA and SA pathways genes.Recombinant hydrophobin protein rHFBII-4 and rHFBII-6 ofT.asperellumcould induce the expression of genes in the auxin signal transduction pathway (Zhang et al.2019; Huang et al.2015).In this study, the separate inoculation of T-Pa2 (Group-T) andA.alternata(Group-A) both altered the expression ofIAA10andGH3.17, showing similar trends of downregulation followed by upregulation, which indicated when PdPap poplar interacts withT.asperellumorA.alternata, the mechanism of auxin signal transduction regulation might be similar.The expression of these two genes under combined inoculation (Group-T + A) showed a different trend.The expression ofGH3.17in Group-T + A was similar to that under separate inoculation; however, the expression ofIAA10in Group-T + A remained downregulated at 7 h and 28 h, in contrast to separate inoculation.Therefore, we inferred that the plants’ response to the microorganisms is complicated and systemic, and when the plants were inoculated with two fungi, the mechanism might be different to that induced by single inoculation.

Conclusion

Herein, nineTrichodermastrains were isolated from a rhizosphere soil ofP.amurens, and identified as three species:T.brevicompactum(one isolate),T.asperellum(two isolates) andT.atroviride(six isolates).T.asperellum-T-Pa2 showed the highest ability to inhibit the growth of six different fungal pathogen species.Furthermore, it increased the CAT activity, NR activity, and content of osmosis molecules in PdPap poplar, and increased the resistance of PdPap poplar againstA.alternatavia JA, SA and auxin signal transduction pathways.The present study provided a robust method to screenTrichodermastrains and initially evaluate their biological control potential, which could be further applied in Northeast China.

AcknowledgementsWe thank Shida Ji, Ruiting Guo, Chang Zhou, Jian Diao, and Ping Zhang for their advice regarding this article.