Solomon Boamah 張樹武 徐秉良 李通 Alejandro Calderón-Urrea

Screening and Identification of Salt-tolerant Trichoderma Species under Salt Stress

Solomon Boamah，ZHANG Shuwu，XU Bingliang，LI Tong and Alejandro Calderón-Urrea

（College of Plant Protection，Gansu Agricultural University/Engineering Laboratory for

Biological Control of Crop Diseases and Pests，Lanzhou 730070，China）

Abstract This study focused on the screening and identification of salt-tolerant Trichoderma species from six strains that were isolated from Minqin County，Gansu Province，China.The results showed that increasing NaCl concentrations（from 0 to 150 mmol/L） reduced the colony diameter in all the six Trichoderma strains（TG1 to TG6）.The strain of TG1 presented higher salt-tolerant ability under different NaCl concentrations in comparison to the other five strains.At 50 mmol/L NaCl-stress，there was no significant difference at the spore concentration and the colony diameter across the two days compared to the control.However，the spore germination of TG1 was increased by 10.59% at 50 mmol/L NaCl? stress，compared to the control.Morphologically，TG1 strain showed dull green mycelial growth on potato dextrose agar medium with slightly dull yellow in? revise colony，conidia were spherical to ellipsoidal，conidiophores having a more or less distinct central axis and five layers of phialides extending across the terminal parts of the conidiophores.According to the morphology of TG1，it was identified as T.longibrachiatum.Phylogenetic analysis based on TEF and ITS revealed that TG1 strain was 99.22% and 100.00% closely related to T.longibrachiatum IMI 297702（EU401578.1） and T.longibrachiatum（MT889709.1），respectively.Thus，TG1 strain was identified as T.longibrachiatum TG1? according to the morphological characteristics and molecular analyses.The activity and concentration of 1-aminocyclopropane-1-carboxylate（ACC-deaminase） and indole-3-acetic acid（IAA） of TG1 strain were increased significantly under salt stress.The IAA concentrations? of TG1 strain were 1.25，? 1.35 and?? 1.56?? μg/mL at 50，100? and 150 mmol/L NaCl stress as compared to the control 0.92 μg/mL，respectively.Similarly，the activities of ACC deaminase of TG1 were 0.1，0.16 and 0.17 μmol α-ketobutyrate mg/h at 50，100，and 150 mmol/L NaCl stress compared to the control 0.09 μmol α-ketobutyrate mg/h，respectively.

Key words Trichoderma spp.; Salt stress; Screening and? identification

Received ?2022-08-22??? Returned 2022-10-17

Foundation item Fuxi Outstanding Talent Cultivation Program，Gansu Agricultural University（No.Gaufx-03J03）; Longyuan Youth Innovation and Entrepreneurship Talent Project（No.2021LQTD27）; National Natural Science Foundation of China（No.31860526）; Gansu Provincial Science Fund for Distinguished Young Scholars（No.18JR3RA161）.

First author Boamah Solomon，male，Ph.D student.Research area：crop protection.E-mail：solomon4408boamah@gmail.com

Corresponding?? author ZHANG Shuwu，male，Ph.D，associate professor.Research area：biological control of plant disease.E-mail：zhangsw704@126.com

XU Bingliang，female，Ph.D，professor.Research area：biological control of plant disease.E-mail：xubl@gsau.edu.cn

Nearly 1 billion hectares of land are affected by soil salinization worldwide.Presently，approximately 1 125 million hectares of lands are salt-affected，of which nearly 76 million hectares are affected by human-induced salinization［1］.Salinity in the soil causes osmotic and ionic stress in plants which affect several important physiological and biochemical processes［2］，i.e.disruption of hormone balance，alteration of protein metabolism，inhibition of the activity of enzymes involved in nucleic acid metabolism，and decrease in chlorophyll［2］.The result of rising land salt stress is now considered? to be a major cause of impaired crop development and poor yields in arid and semi-arid areas，and the number of land affected by salt stress is increasing internationally，which is a cause for concern［3］.Salt-tolerant crops are being developed using genetic engineering and molecular marker-assisted breeding methods，but biocontrol technique，such as the use of salt-tolerant plant growth-promoting rhizobacteria（PGPR），is also gaining attention in modern agricultural system［4-6］.Plant growth-promoting microbes（PGPM） possess several potentials that enhance plant growth by a wide variety of mechanisms such as production of 1-aminocyclopropane-1-carboxylate deaminase（ACC-deaminase），indole-3-acetic acid（IAA），phosphate solubilization，siderophore production and nitrogen fixation［7］.In addition，they indirectly promote plant growth by controlling pathogens through competition for nutrients and space，secretion of antibiotic substances，and induction of defence systems in the plant［8］.Consequently，the use of PGPR as elicitors for the plant “induced systemic tolerance” to abiotic stimuli ［9-10］，such as salt stress and solution for plant salt stress management has been recommended.

Trichoderma species are plant growth-promoting fungi that play an important role in alleviating abiotic and biotic stresses due to their antimicrobial，mycoparasitic，competitive and secondary metabolic potentials，antioxidant enzyme activity and gene expression，and synthesis of phytohormones such as IAA，auxins，cytokinins，ethylene，gibberellins（GAs），and abscisic acid（ABA）［9-10］.Likewise，several fungi have the considerable ability to produce certain enzymes such as ACC-deaminase produced by T.longibrachiatum T6（TL-6） which involved in promoting wheat? growth and enhancing plant tolerance to NaCl stress［11］.T.asperellum T203 produces ACC-deaminase that regulates the endogenous ACC level and stimulates root elongation and enhances plant tolerance to abiotic stress［12］.To date，researches have mostly focused on identification of salinity tolerant strains of Trichoderma and very few reports are available on salt tolerant Trichoderma species capable of producing both IAA and ACC-deaminase under salinity stress by? in vitro studies.Therefore，this study aims to screen and identify the potential of salt-tolerant Trichoderma strain that can produce both IAA and ACC-deaminase under salinity stress.The results will be important for further research to determine the mechanisms by which the strain exerts its salt-tolerant properties to facilitate its future uses，especially in arid and semi-arid area for soil salinity management.

1 Materials and methods

1.1 Fungal inoculum preparation

Six Trichoderma strains were collected from the corn，chili and sunflower rhizosphere soil in Minqin county，Gansu Province，China and stored in the Laboratory of Plant Pathology at Gansu Agricultural University.All strains were cultured on potato dextrose agar（PDA） in Petri dishes for 7 days at 25 ℃.The colony morphology of each strain was recorded and named according to the numbers from TG1 to TG6.

1.2 Salt concentration preparation

The salt tolerance assay for the strains was performed according to themethod described by Zhang et al.［13］.The final NaCl concentrations of PDA media were prepared as 0，50，100，and 150 mmol/L.

1.3 Assessment of mycelial growth of Trichoderma strains under different salt concentrations

All selected Trichoderma strains were screened for salinity tolerance by inoculating 5 mm of mycelial disc of each strain separately on PDA medium supplemented with 0，100 and 150 mmol/L NaCl and incubated at 25 ℃.Out of 6 strains，one of the tolerant strains was selected on the basis of its growth at 0，50，100 and 150 mmol/L.This was further screened for salinity tolerance at different NaCl concentrations.The colony growth at given salinity was assessed daily by measuring the diameter of the Trichoderma strain［14］.This was repeated three times for each strain.The inhibition rate was calculated following the formula;

Inhibition rate=（Average diameters of the control-Average diameters of the treatment）/（Average diameters of the control-0.5）×100%

Where control represents samples without NaCl stress and treatment represents samples with NaCl stress，0.5 represents the diameter of the mycelial disc.

1.4 Determination of spore concentration and germination

The strain TG1 spore suspension of 1.0×108 spores/mL was prepared according to Zhang et al.［15］.The spore concentration in the suspension was determined with a haemocytometer，and the concentration was prepared and adjusted to?? 1×108 spores/mL.To determine? the spores germination，TG1 spore suspension was spread on the surface of water agar in Petri dishes.Petri dishes were sealed with Parafilm and incubated at 25 ℃ for 24 h，and each treatment and control were repeated three times.Three disks from each replicate agar plate were cut out with a cork borer and placed on a slide.A total of 50 spores per disk（150 per agar plate） was counted using microscope.Spores were considered to have germinated when the germ-tube length was equal to or greater than the radius of the spore［16］.

1.5 Morphological identification

The morphological characteristics of the selected strain（TG1），such as the morphology of the colony and spores，were examined for 5 days at 25 ℃ after culture on PDA media.Microscopic examination and measurement of conidiophores，phialides，and conidia were carried out according to the method of Shah et al.［17］.A total number of 100 conidia were? used to determine the conidial morphology under?? 400× magnification with the automated inverted bright-field microscope（Leica DMI6000 B）［20］.The morphological identifications was performed following Castle［18］ and Bissett［19］ mode of morphological classification of Trichoderma species.

1.6 Molecular identification

Extraction and purification of total genomic DNA were carried out according to the method of Yoshinaga et al.［21］.The extracted DNA from the selected strain TG1 was used as a template for polymerase chain reaction（PCR） based on the amplification of the internal transcribed spacer（ITS） and translational elongation factor gene（TEF）.Amplification was performed?? using primers were listed in Table 1.PCR amplification of DNA was performed with the following settings：initial denaturation 5 min at 95? ℃，30 cycles of 30 s at 95? ℃，30 s at 54? ℃ and 1 min at 72? ℃ and final extension for 7 min at 72? ℃ for the ITS.TEF? PCR was set for 3 min at 97? ℃，35 cycles of 30 s at 96? ℃，1 min at 58? ℃ and?? 1 min at 72? ℃ and final extension for 10 min at 72? ℃.The amplified products were analysed by agarose gel electrophoresis.The best-amplified products were sent to TSINGKE Biological Technology Company（Xian，China） for automated DNA sequencing.Partial sequences obtained with forward and reverse primers were combined into full-length sequences and compared with sequences in the GenBank database of the National Center for Biotechnology Information（NCBI）（http：//www.ncbi.nlm.nih.gov/GenBank） using the Basic Local Alignment Search Tool（BLAST） to identify close phylogenetic relatives.The sequences of ITS and TEF were?? aligned together with reference sequences，and a neighbour-joining analysis was used to construct a phylogenetic tree using MEGA X.10 software［22］.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test（1 000 replicates） is indicated next to the branches.

Table 1 Primers for Trichoderma strain identification

表1 木霉菌株鑒定引物

Target gene目標(biāo)基因Sequence序列（5′→3′）

ITSF：TCCGTAGGTGAACCTGCGG

R：TCCTCCGCTTATTGATATGC

TEFF：CATCGAGAAGTTCGAGAAGG

R：TACTTGAAGGAACCCTTACC

1.7 Indole acetic acid（IAA） and 1-aminocyclopropane-1-carboxylate deaminase（ACCD） production

1.7.1 Indole acetic acid（IAA） Indole-3-acetic acid（IAA） was determined according to the method of Fu et al.［23］.HPLC analysis of IAA was performed on a C18 column（5 μm; 25×? 0.46 cm） using an HPLC system of acetonitrile and?? 0.1% trifluoroacetic acid，programmed over 14 min at a flow rate of 10?? μL/min with a UV detector at 220 nm and??? 40 ℃.The mobile phase consisted of methanol and water（80∶20 v/v） run at the flow rate analysed by comparison with the elution profiles of the standard IAA.

1.7.2 1-aminocyclopropane-1-carboxylate deaminase（ACCD） 1-aminocyclopropane-1-carboxylate deaminase（ACCD） was inoculated from 1 mL of spore suspension of TG1（1×108 spores mL-1） in 60 mL of a synthetic medium containing 0，50，100，and 150 mmol/L NaCl.The?? amount of ACC produced was measured by the amount of α-ketobutyrate produced，and expressed as μmol α-ketobutyrate mg/h［24］.

2 Results and analysis

2.1 Screening of NaCl-tolerant Trichoderma strains

Increasing NaCl concentration significantly（P<0.05） decreased colony diameter in all six Trichoderma strains（Table 2）.The concentration of 100 mmol/L NaCl stress on day 1 decreased the diameter growth of six strains by?? 2.78%，20.10%，18.54%，37.65%，40.63% and 22.45% compared to the respective controls of TG1-TG6. Except strain TG1 which had no significance difference（P>0.05） with the control（0 mmol/L NaCl） on day 1 under 100 mmol/L NaCl stress（Table 2）.At 150 mmol/L NaCl stress on day 1，the diameters of the strains were decreased by 21.53%，47.94%，50.99%，? 54.32%，60.94% and 41.33% compared to their respective controls.From TG1-TG6 on day 2，100 mmol/L NaCl stress decreased the diameter of the strains by 3.02%，5.59%，12.01%，? 8.41%，12.53% and 6.16% compared to the respective controls; and 150 mmol/L NaCl stress decreased the diameter growth of the strains by 30.65%，38.22%，72.94%，49.04%，56.16%，and 49.05% compared with the respective controls（Fig.1）.Therefore，Trichoderma strain TG1 was selected for further characterisation as it showed best growth under salinity stresses? compared to other strains.

2.2 Re-evaluation of TG1 under salt stress

Salt stress affected colony growth，spore concentration，and germination of TG1 strain with the changes of NaCl? concentrations.An increase in NaCl concentration increased the inhibitory rate of TG1 colony growth at days 1 and 2.Compared to control，50，100 and 150 mmol/L NaCl stress exhibited TG1 colony growth inhibition by 6.95%，2.98% and 14.57% at day 1;?? 0.38%，3.67% and 6.07% at day 2，respectively.Similarly，50，100 and 150 mmol/L NaCl stress exhibited inhibition of TG1 spore concentration by 3.12%，10.58% and 13.03%，respectively at day 7 compared to control.However，at 50，100 and 150 mmol/L NaCl stress，the spore germination rate increased by 10.59%，? 1.67% and 0.10% respectively compared to control（Table 3）.Therefore，TG1 strain exhibited best characterisation at 50 mmol/L NaCl stress after re-evaluation.

2.3 Morphological identification of strain TG1

The front colony of TG1 showed dull green mat anterior color on PDA medium（Fig.2-A） and the revise colony showed slightly dull yellow color（Fig.2-B）.The phialides occur singly?? along the major axis of conidiophores and branched from the major axis（Fig.2-C）.The standard conidiophores have a more or less distinct central axis with single phialides extending across the terminal part and branching occurring within about five layers of phialides（Fig.2-C）.The length of the lateral branches increases with distance from the apex，and like the main axis，they contain solitary phialides（Fig.2-C）.The conidia? of? TG1 were spherical to ellipsoidal with an average conidia length of 3 to 5 μm and width of?? 2.5 to 3 μm（Fig.2-D）.The strain TG1 was classified as T.longibrachiatum based on its discernible morphological characters.

2.5 IAA and ACC-deaminase synthesis of TG1 under NaCl stress

The IAA concentration and ACC deaminase of TG1 were increased significantly（P<0.05） at different NaCl concentrations（0，50，100，and 150 mmol/L）.The IAA concentrations of the TG1 were 1.25，1.35 and 1.56 μg/mL at 50，100，and 150 mmol/L NaCl stress as compared to the control 0.92 μg/mL，respectively（Fig.4-A）.Similarly，at 50，100，and 150 mmol/L NaCl stress，the activities of ACC-deaminase of TG1 were 0.1，0.16，and 0.17 μmol α-ketobutyrate mg/h compared to the control 0.09 μmol α-ketobutyrate mg/h，respectively（Fig.4-B）.

3 Discussion

The present study aimed at screening and?? identification of Trichoderma strains with high salinity tolerance and evaluate for further characterisation of IAA and ACC-deaminase production.Our findings showed? that? the salinity stress restricts the colony growth of the Trichoderma? strains in PDA medium［25］.The mycelia growth of the Trichoderma strains varied in response to the increase of NaCl concentrations.However，the TG1 strain showed a lower inhibition rate caused by the NaCl stress with dense mycelia growth of colonies compared to the other strains.The relative high inhibition rate of strains TG2 to TG6 may indicate intolerance to increasing salinity in the growth medium.These results are consistent with previous studies on fungi by Dixon et al.［26］and Hutchison［27］ in which NaCl stress decreased the growth rate of some fungi，while other strains were found to be tolerant.Again，the inhibition rate of the spore concentration of strain TG1 was lower compared to the other strains under the NaCl stresses.Furthermore，50 mmol/L NaCl stress increased the spore germination compared to the control.These finding are in agreement to that of Mandeel［28］，who reported that at greater salinities，sporulation increased and fewer hyphae were observed.Hujslová et al.［29］ and Nayak?? et al.［30］ also reported that most fungal isolates from hypersaline natural habitats are halotolerant rather than halophilic，with many thriving better at lower salinities.

Previous studies on Trichoderma identification were based on morphology and either the use of internal transcribed spacer（ITS） or translational elongation factor（TEF） sequence analysis.Rinu et al.［31］ who reported that T.gamsii was identified based on phenotypic（colony morphology and microscopy） and 18S rDNA sequence（ITS5） analysis.Again，Rai et al.［32］?? identified the Trichoderma isolates according to morphology and based on the translation elongation factor 1-α gene sequence（TEF） similarity，the isolates were designated as T.harzianum，T.koningii，T.asperellum，T.virens and T.viride.However，the identification of the Trichoderma strain TG1 as T.longibrachiatum in our study was based on colony morphology and amplification of both ITS? and TEF sequence?? analysis. In agreement with our study，Azriah?? et al.［33］ also identified Trichoderma strains based on its morphological characteristics and using both （ITS） 1 and 2? and? TEF-1α? genes.

PGPM? possess several potentials that enable them to enhance plant growth.These PGPM potentials include the production of phytohormones such as IAA which help in stress tolerance.The strain T.longibrachiatum TG1 produced both IAA and ACC-deaminase under NaCl stress compared to control.The increased ACC-deaminase and IAA concentration in the TG1 strain may serve as an important signal to alleviate the negative effect of NaCl stress on the fungi growth.In this study，the IAA and ACC-deaminase production in the TG1 under salt stress depends on the concentration of NaCl; an increase in NaCl concentration from 50 to? 150 mmol/L increased the IAA and ACC-deaminase production significantly.In this regard，ACC-deaminase produced by TG1 may acts as a biological sink for ACC.This is consistent with the findings of Kang et al.［34］ where Leclercia adecarboxylata（MO1） producing ACC deaminase and IAA improved salinity tolerance.It is also consistent with previous studies where majority of microbes reported facilitating phytoremediation of metals synthesize both ACC deaminase and IAA［35］.In our? and other studies，the increased level of the IAA and ACC-deaminase in the beneficial fungi under NaCl stress may serve as an important signalling for salinity tolerance［36］.

4 Conclusions

In the present study，the Trichoderma strain TG1 had a great potential to tolerate salinity stress compared to the other strains.Based on colony morphology，microscopic features and amplification of? ITS? and TEF genes sequence analysis，Trichoderma strain TG1 was identified as T.longibrachiatum.Trichoderma? longibrachiatum? TG1 was also found to produce both IAA and ACC-deaminase under salinity stress.

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鹽脅迫下耐鹽性木霉菌的篩選與鑒定

Solomon Boamah，張樹武，徐秉良，李 通，Alejandro Calderón-Urrea

（甘肅農(nóng)業(yè)大學(xué)? 植物保護(hù)學(xué)院/甘肅省農(nóng)作物病蟲害生物防治工程實(shí)驗(yàn)室，蘭州 730070）

摘 要 對(duì)分離于甘肅省民勤縣的6株木霉菌進(jìn)行耐鹽性篩選和鑒定。結(jié)果表明，隨著NaCl 濃度（0 to 150 mmol/L） 增加顯著降低6株木霉菌（TG1 to TG6） 的菌落直徑。與其他5株木霉菌相比，不同濃度NaCl脅迫下菌株TG1具有較強(qiáng)耐鹽性。當(dāng)NaCl濃度為 50 mmol/L 時(shí)，培養(yǎng)2 d后菌株TG1產(chǎn)孢量和菌落直徑與對(duì)照相比無顯著差異，但是其孢子萌發(fā)率增加10.59%。菌株TG1在PDA培養(yǎng)基上菌落正面呈暗綠色，菌落背面呈暗黃色，分生孢子球形至橢圓形。分生孢子梗直角分枝，結(jié)合其形態(tài)特征將菌株TG1鑒定為T.longibrachiatum。構(gòu)建菌株TG1的TEF和ITS系統(tǒng)發(fā)育樹，結(jié)果表明菌株TG1的TEF和ITS序列分別與T.longibrachiatum IMI 297702（EU401578.1）和T.longibrachiatum（MT889709.1） 序列相似性為99.22%和100.00%。因此，結(jié)合形態(tài)特征和分子生物學(xué)分析將菌株TG1鑒定為T.longibrachiatum TG1。與對(duì)照相比，菌株 TG1 在 50、100 和 150 mmol/L NaCl 脅迫下產(chǎn)生IAA濃度分別為 1.25、1.35和 1.56 μg/mL，而對(duì)照為0.92 μg/mL。同時(shí)，在 50、100 和 150 mmol/L NaCl 脅迫下，TG1 菌株ACC脫氨酶活性分別為 0.1、0.16和 0.17 μmol? α-ketobutyrate mg/h，而對(duì)照為0.09 μmol α-ketobutyrate mg/h。

關(guān)鍵詞 木霉；鹽脅迫；篩選和鑒定

收稿日期：2022-08-22? 修回日期：2022-10-17

基金項(xiàng)目： 甘肅農(nóng)業(yè)大學(xué)“伏羲杰出人才培育計(jì)劃”項(xiàng)目（Gaufx-03J03）；隴原青年創(chuàng)新創(chuàng)業(yè)人才項(xiàng)目（2021LQTD27）；國家自然基金（31860526）；甘肅省杰出青年基金（18JR3RA161）。

第一作者：Boamah Solomon，男，在讀博士，研究方向?yàn)樽魑锉Ｗo(hù)。E-mail：solomon4408boamah@gmail.com

通信作者：張樹武，男，博士，副教授，研究方向?yàn)橹参锊『ι锓乐巍-mail：zhangsw704@126.com

徐秉良，女，博士，教授，研究方向?yàn)橹参锊『ι锓乐?。E-mail：xubl@gsau.edu.cn

（責(zé)任編輯：顧玉蘭 Responsible editor：GU Yulan）