Spore production in the solid-state fermentation of stevia residue by Trichoderma guizhouense and its effects on corn growth

LIU Hong-jun,DUAN Wan-dong,LIU ChaoMENG Ling-xueLI Hong-xuLI RongSHEN Qi-rong

1 Jiangsu Provincial Key Lab of Solid Organic Waste Utilization/Key Laboratory of Plant Immunity,Jiangsu Collaborative Innovation Center of Solid Organic Wastes/Educational Ministry Engineering Center of Resource-saving fertilizers/Nanjing Agricultural University,Nanjing 210095,P.R.China

2 Institute of Botany,Jiangsu Province and Chinese Academy of Sciences,Nanjing 210014,P.R.China

Abstract Trichoderma is an important and widely used plant growth-promoting fungus (PGPF).In this study,stevia residue amended with amino acids hydrolyzed from animal carcasses was used for the production of Trichoderma guizhouense NJAU 4742 by solid-state fermentation,and then its potential to promote corn plant growth was evaluated in combination with chemical fertilizer (CF) or organic fertilizer (OF).The highest spore number of 7×109 CFU g–1 fresh weight was obtained under the following optimal parameters:material ratio of 50% (stevia residue:rice bran=1:1),pH value of 3.0 (amended with 6.67% amino acids),initial moisture content of 60%,inoculum size of 10%,material thickness of 3 cm and an incubation time of 4 days.The aboveground corn plant biomass obtained with T.guizhouense applied alone and with CF treatments were slightly higher than those of no fertilizer control and CF treatments,respectively.However,T.guizhouense applied with OF significantly(P<0.05) increased aboveground biomass compared to OF and yielded the highest aboveground biomass among all the treatments.Moreover,T.guizhouense applications primarily influenced the fungal bulk soil community composition,among which three OTUs (OTU_2 and OTU_9 classified as Chaetomium,and OTU_4 classified as Trichoderma) were stimulated in both bulk and rhizosphere soil.Notably,a specific OTU_3 (Phymatotrichopsis) was only stimulated by T.guizhouense applied with OF,possibly leading to high soil productivity.These results show that it is feasible to employ stevia residue in the eco-friendly fermentation of T. guizhouense,which is strongly suggested for enhancing OF applications.

Keywords:Trichoderma,solid-state fermentation,stevia residue,plant growth-promoting fungi,soil fungal community

1.Introduction

The use of plant growth-promoting fungi (PGPFs) is considered a promising alternative to chemical fertilizers and pesticides,which cause severe damage to the environment(Harmanet al.2004).Among the PGPFs,it is well recognized thatTrichodermaspp.can effectively promote plant growth (Shoreshet al.2010).Specifically,Trichodermaspecies display plant growth-promoting versatility,as they can produce phytohormones,decompose organic matter,and protect plants from biotic and abiotic stresses (Caiet al.2015;Druzhininaet al.2018).However,successful plant promotion requires the application of abundant spores ofTrichodermainto soil (Zhanget al.2016).Consequently,it is crucial to obtain a high density ofTrichodermaspores.

Solid-state fermentation (SSF) is a biotechnological process that involves the processing of solid substrates in the absence or near absence of free water (Pandeyet al.2000).The use of SSF is well-studied for the production of enzymes,such as cellulases,lipases,and chitinases (De la Cruz Quirozet al.2015).Compared to liquid-state fermentation,SSF is closer to the natural habitat and many species successfully produce spores through SSF (Singhaniaet al.2009).Moreover,the fungal spores produced by SSF have a longer shelf life than those produced by liquid-state fermentation (Vicciniet al.2007).For the SSF ofTrichoderma,solid materials like wheat bran,sorghum pulp,coffee or tea wastes,and sugarcane bagasse have been used as substrates,but the low spore yields limitTrichodermacommercialization at a larger scale (Rayhaneet al.2019).Thus,it is crucial to seek a more suitable raw material for the growth and sporulation ofTrichoderma.

Stevia rebaudianais a shrub belonging to the Asteraceae family whose leaves contain high concentrations of natural sweeteners (Yokoyamaet al.2019).Subsequent to the extraction of natural sweeteners,a large quantity of solid waste (stevia residue) is produced,which can result in serious environmental issues if not effectively managed(Cobanet al.2012).In addition,stevia residue contains abundant fiber and protein,which may be suitable for SSF ofTrichoderma.There are some other parameters that affect the efficiency of SSF,such as incubation time,incubation temperature,initial moisture content,inoculum size,initial pH,and additional nutrition sources (Nampoothiriet al.2004;Mekalaet al.2008).Similarly,initial moisture contents and inoculum levels are also routinely tested for the optimization of spore production (Vicciniet al.2007).Therefore,it is essential to optimize the conditions for spore production byTrichodermaspp.in the SSF of stevia residue.

Trichodermaspp.are used all over the world as biofertilizer fungi (Cotxarreraet al.2002;Caiet al.2015;Chenet al.2019).Currently,the most widely practiced method to exploitTrichodermaspp.is the application of the fungi on plants or as soil amendment directly (Chenet al.2019).For example,tomato seeds treated withT.koningiihad a stronger ability to suppress soil pathogens than the untreated control (Tsahouridou and Thanassoulopoulos 2002).The combined application ofT.harzianumand chemical fungicides had the ability to control southern corn leaf blight disease (Wanget al.2019).The application ofT.asperellummixed with diatomaceous earth and ground wheat bran significantly increased maize yield in a field trial (Heet al.2019).Rhizosphere colonization is a key requirement for the application of beneficial microorganisms(Caoet al.2020).AlthoughTrichodermaspp.are widely welcomed by farmers for plant promotion,their application is often challenged by the stresses of field conditions,thereby leading to occasionally unpredictable effects (Legrandet al.2017).Therefore,it is necessary to evaluate the effects ofTrichodermaapplication under different treatment strategies.

The aim of this study was to optimize the SSF conditions for the production ofT.guizhouenseNJAU 4742 spores using stevia residue as a substrate.Moreover,theT.guizhouensespores produced were further employed to evaluate their effects on corn grown in combination with either chemical fertilizer or organic fertilizer.The primary objectives of this study were:1) to assess the suitability of stevia residue for SSF ofT.guizhouense;2) to evaluate the effects ofT.guizhouensespores on corn growth;and 3) to identify dynamic changes in the fungal communities followingTrichodermaapplications based on a highsequencing platform.

2.Materials and methods

2.1.Microorganism and culture conditions

TrichodermaguizhouenseNJAU 4742 (China General Microbiology Culture Collection Center Accession no.3308)was supplied by the Jiangsu Provincial Key Lab of Organic Solid Waste Utilization,Nanjing,China.The preparation ofT.guizhouensesuspension (with mycelium and spores)was performed according to Caiet al.(2015) with minor modifications.In brief,T.guizhouensewas cultured on potato dextrose agar (PDA) at 28°C for 7 d and preserved at 4°C on plates.TheT.guizhouensespore suspension was then prepared by flooding PDA medium plates with sterile water and filtering through a double layer of sterile cheesecloth.The number of spores was adjusted to 108CFU mL–1based on hemocytometer counts.The spore suspension was finally inoculated into PDA liquid medium(1%,v/v) and cultivated at 170 r min–1at 28°C for 4 d to obtainT.guizhouensesuspension (with mycelium and spores).

2.2.Optimization of solid-state fermentation process parameters

Stevia residue,rice bran,wheat bran,rice chaff and rice straw powder were collected from a local farm,and their properties are shown in Appendix A.In addition,compound liquid amino acids were preparedviathe acidic hydrolysis of pig corpses by Lianye Co.,Ltd.,Jiangyin City,Jiangsu Province,China.The amino acid preparation contained H+and free amino acid concentrations of 5.23 mol L–1and 124.16 g L–1,respectively.Stevia residue was used to conduct various single-parameter optimization studies for obtaining the maximum spore production byT.guizhouensein solid-state fermentation.This approach included a combined material selection (rice bran,wheat bran,rice chaff,rice straw powder),material ratio of stevia residue and rice bran (10,30,50,70 and 90%,w/w),fermentation thickness (1,2,3,4 and 5 cm),initial moisture content (50,55,60,65 and 70%),T.guizhouensesuspension incubation size (5,7.5,10,12.5 and 15%,v/w),and fermentation time (2,3,4,5 and 6 d).In our previous study,the optimal pH value of 3 was found forT.guizhouensestrain spore production in SSF (Miaoet al.2020).Thus,all the media were adjusted to pH 3.0 by adding hydrolytic amino acids (appending proportion 6.67%,v/w),autoclaved at 115°C for 40 min and then cooled to 28°C.Three sets of independent experiments were performed for each parameter optimization.

2.3.Pot experimental design and soil sampling

A two-season pot experiment was conducted in a greenhouse located at the Jiangsu Key Lab for Solid Organic Waste Utilization,Yixing,China,from April to June 2018 and April to June 2019.The pot experiment included six treatments:(1) soil amended with no fertilizer (CK);(2) soil amended withT.guizhouense(CK+Tri.),(3) soil amended with chemical fertilizer (CF),(4) soil amended with chemical fertilizer plusT.guizhouense(CF+Tri.),(5) soil amended with mature cow manure compost (OF),and (6) soil amended with mature cow manure compost plusT.guizhouense(OF+Tri.).Mature cow manure compost was produced by Jiangyin Lianye Co.,Ltd.,Jiangsu,China,by composting cow manure at 50–70°C for more than 15 d.Corn was used as a model plant,and each treatment included six pots.The application amount of organic fertilizer was 60 g DW per pot,and solidT.guizhouense spores was applied at 6 g fresh weight (FW) per pot.The CF-containing treatments received the same levels of nitrogen (urea),phosphorus(calcium superphosphate) and potassium (K2SO4) as the OF-containing treatments.Forty days after corn plantlet transplantation into pots,plant biomass was determined.Soil sampling was performed in June 2019 during plant biomass measurement.Briefly,four plants were carefully removed at random for each treatment.The bulk and rhizospheric soil samples were obtained according to Liuet al.(2017).

2.4.DNA extraction and Illumina MiSeq sequencing

Total DNA from each soil sample was extracted using Power Soil DNA Isolation Kits (MoBio Laboratories,Carlsbad,CA,USA) according to the manufacturer’s protocol.The quality and concentration of the extracted DNA samples were determined by a NanoDrop 2000 spectrophotometer(Thermo Scientific,USA).All DNA samples served as the templates for the amplification of the ITS region with the primers ITS1F (5´-CTTGGTCATTTAGAGGAAGTAA-3´)and ITS2 (5´-GCTGCGTTCTTCATCGATGC-3´) according to Huanget al.(2015).The programs of amplification and sequencing of the ITS genes were performed at Personal Biotechnology Co,Ltd.(Shanghai,China) on an Illumina MiSeq instrument (USA).The barcode information of the samples is shown in Appendix B.All sequences were submitted in the NCBI Sequence Read Archive database with the accession number of PRJNA644667.

2.5.Bioinformatic analysis

The quality control analysis of the original sequences of samples was performed using UPARSE (Edgar 2013).The forward and reverse sequences obtained directly from the company were merged,and then quality control of the merged sequences was conducted.High-quality sequences were obtained by removing sequences withq-values greater than 0.5,lengths less than 200 bp,repetitive sequences and single sequences.Then,USEARCH was used to cluster high-quality sequences into operational taxonomic units (OTUs,97% similarity).Finally,the representative sequences of each OTU were classified in the UNITE database (Kõljalget al.2013).In addition,to compare the fungal microbial community structures of all soil samples,principal coordinate analysis (PCoA) was accomplished at the OTU level based on the Bray-Curtis distance metric using MOTHUR (Schlosset al.2009).Stamp analysis was performed to find the response OTUs that were stimulated byTrichoderma-amended treatments.Moreover,Pearson’s correlation coefficient was used to evaluate the correlations between the relative abundances of the response OTUs in bulk and rhizosphere soil samples.

2.6.Statistical analysis

The pot experiment was repeated twice,and a similar trend was observed.Thus,the data from April to June 2019 are presented.The differences among the treatments were assessed using one-way ANOVA,and the calculated means were subjected to Duncan’s multiple range test atP<0.05.All analyses were performed in IBM SPSS Statistics 22(SPSS Inc.,Chicago,IL,USA).

3.Results

3.1.SSF and optimization of culture conditions

The effects of different parameters onT.guizhouensespore production in SSF are shown in Fig.1.The results showed that stevia residue amended with rice bran exhibited the highest spore production compared to the other treatments(Fig.1-A).The optimal material ratio of stevia residue and rice bran was found to be 1:1 (Fig.1-B).While there was no significant difference in the cell density between 2 and 3 cm of the material thickness,the spore production abilities of these two treatments were higher than those of the other treatments (Fig.1-C).Moreover,the optimal initial moisture content of SSF was 60% (Fig.1-D).For inoculum size,the cell density increased with an increase in the inoculation amount until 10%,but no significant difference was observed among the 10,12.5 and 15% treatments (Fig.1-E).Similarly,the cell density increased with increasing incubation time until the 4th day (Fig.1-F).In summary,the optimal parameters of the solid-state fermentation process were a material ratio of stevia residue to rice bran of 1:1,a material thickness of 3 cm,an initial moisture content of 60%,an inoculum size of 10% and an incubation time of 4 d.Notably,the maximum number of spores reached 7×109CFU g–1FW.

3.2.Effects of different treatments on corn growth in the pot experiment

The effect of different treatments on corn growth in the pot experiment is shown in Fig.2.The underground biomass of the CK+Tri.,CF+Tri.and OF+Tri.treatments were slightly lower (P>0.05) than those of CK,CF and OF treatments,respectively.In contrast,the aboveground biomass of the CK+Tri.treatment was slightly higher (P>0.05) than that of the CK treatment,as well as the CF+Tri.treatment compared to the CF treatment.Moreover,the OF+Tri.treatment significantly (P<0.05) increased aboveground biomass compared to the OF treatment.Furthermore,the OF+Tri.treatment had the highest aboveground biomass among all the treatments.

3.3.Effects of different treatments on fungal alpha diversity

Fig.1 Effects of different parameters on Trichoderma guizhouense NJAU 4742 in solid-state fermentation of stevia residue.SR,stevia residue;RB,rice bran;B,bran;RC,rice chaff;RSP,rice straw powder.Different letters above the error bars (SD) denote significant differences at the 0.05 probability level according to Duncan’s multiple range test (n=3).

Fig.2 Effects of different fertilizer treatments on corn growth.CK,no fertilizer;CK+Tri.,Trichoderma guizhouense NJAU 4742(Tri.) applied with CK;CF,chemical fertilizer;CF+Tri.,Tri.applied with CF;OF,organic fertilizer;OF+Tri.,Tri.applied with OF.Different letters above (lowercase) and below (uppercase) the error bars (SD) denote significant differences for aboveground and underground biomass,respectively,at the 0.05 probability level according to Duncan’s multiple range test (n=3).

The effect of different treatments on fungal alpha diversity is shown in Fig.3.There was no significant difference among all the treatments in the rhizosphere fungal Chao1 index (Fig.3-A).However,theTrichoderma-amended treatments CK+Tri.and CF+Tri.significantly reduced the bulk soil fungal Chao1 index compared to the CK and CF treatments,respectively.Moreover,the OF+Tri.treatment also decreased the fungal bulk Chao1 index compared to the OF treatment,although no significant difference was observed.Similarly,there were no significant differences of the fungal Shannon index in the rhizosphere between CK and CK+Tri.treatments or between CF and CF+Tri.treatments or between OF and OF+Tri.treatments (Fig.3-B).However,theTrichoderma-amended treatments CK+Tri.,CF+Tri.and OF+Tri.significantly reduced the bulk soil fungal Shannon index compared to those in the CK,CF and OF treatments.

3.4.Fungal community composition

PCoA based on the Bray-Curtis distance metric revealed that the bulk soil fungal communities differed significantly(R2=0.385,P<0.001) from those in the rhizosphere along with the first component (Fig.4).For bulk soil,theTrichodermaamended treatments CK+Tri.,CF+Tri.and OF+Tri.were well separated from the CK,CF and OF treatments along with the second component.However,no clear difference in the composition of the rhizosphere fungal community among all the treatments was observed.In addition,treatments(R2=0.232,P<0.001) and the sites×treatments (R2=0.149,P<0.01) both significantly influenced the fungal community composition.

3.5.Responsive OTUs

Stamp analysis of abundant OTUs (relative abundance more than 0.5%) between the CK and the CK+Tri.treatments or between CF and CF+Tri.or between OF and OF+Tri.in the bulk and rhizosphere soil are presented in Appendix C.Compared to the CK treatment,the CK+Tri.treatment significantly increased the relative abundances of OTU_2,OTU_4,OTU_9,OTU_11,OTU_19 and OTU_45 in the bulk soil (Appendix C).Moreover,the CF+Tri.treatment significantly increased the relative abundances of OTU_2,OTU_4,OTU_9,OTU_23,OTU_11,OTU_15,OTU_40,OTU_19 and OTU_30 in bulk soil compared to the CF treatment (Appendix C).Furthermore,the OF+Tri.treatment significantly increased the relative abundances of OTU_2,OTU_4,OTU_9,OTU_23,OTU_12,OTU_3,OTU_11,OTU_52 and OTU_64 in bulk soil compared to the OF treatment (Appendix C).The overlapping OTUs of the above responsive OTUs induced byTrichodermaapplications were OTU_2,OTU_4,OTU_9 and OTU_11 in the bulk soil (Fig.5-A).Moreover,the solitary OTUs in OFvs.OF+Tri.but not in other pairwise comparisons in the bulk soil were OTU_3,OTU_12,OTU_52 and OTU_64.For the rhizosphere soil,the CK+Tri.treatment significantly increased the relative abundances of OTU_2,OTU_4,OTU_9,OTU_10,OTU_16,OTU_28,OTU_26,OTU_18,OTU_6,OTU_49,OTU_3,OTU_8 and OTU_56 compared to the CK treatment (Appendix C).Moreover,the CF+Tri.treatment significantly increased the relative abundances of OTU_2,OTU_4,OTU_9,OTU_10,OTU_3,OTU_232,OTU_18 and OTU_60 compared to the CF treatment (Appendix C).Furthermore,the OF+Tri.treatment significantly increased the relative abundances of OTU_2,OTU_4,OTU_9,OTU_232,OTU_10,OTU_14,OTU_3 OTU_8,and OTU_13 in bulk soil compared to the OF treatment (Appendix C).Thus,the overlapping OTUs of the responsive OTUs induced byTrichodermaapplications in the rhizosphere soil were OTU_2,OTU_4,OTU_9,OTU_3 and OTU_10 (Fig.5-B).Interestingly,the responsive OTU_2 (Chaetomium),OTU_4 (Trichoderma),and OTU_9(Chaetomium) appeared in both bulk and rhizosphere soils (Appendix D).In particular,the relative abundances of all three of these OTUs in bulk soil had significant and positive relationships with the corresponding OTUs in the rhizosphere soil (Fig.5-C and E).Notably,the responsive OTU_3 (Phymatotrichopsis) that was stimulated in the rhizosphere soil byT.guizhouenseapplication was only induced by the OF+Tri.treatment in the bulk soil.

Fig.3 Effects of different treatments on the fungal Chao1 index (A) and Shannon index (B) in bulk and rhizosphere soil.CK,no fertilizer;CK+Tri.,Trichoderma guizhouense NJAU 4742 (Tri.) applied with CK;CF,chemical fertilizer;CF+Tri.,Tri.applied with CF;OF,organic fertilizer;OF+Tri.,Tri.applied with OF.Different lowercase and uppercase letters above the box plot denote significant differences for bulk and rhizosphere soil,respectively,at the 0.05 probability level according to Duncan’s multiple range test (n=3).

Fig.4 Fungal microbial community compositions of the different treatments in bulk and rhizosphere soil.CK,no fertilizer;CK+Tri.,Trichoderma guizhouense NJAU 4742 (Tri.) applied with CK;CF,chemical fertilizer;CF+Tri.,Tri.applied with CF;OF,organic fertilizer;OF+Tri.,Tri.applied with OF.Differences in the fungal beta diversities were determined by analysis of molecular variance (AMOVA,**,P<0.01 and ***,P<0.001).

Fig.5 Overlap of the responsive operational taxonomic units induced by Trichoderma applications in bulk (A) and rhizosphere (B)soil and Spearman’s rank correlation coeffcients of the relative abundance between bulk and rhizosphere of OTU2 (C),OTU4 (D)and OTU9 (E).CF,chemical fertilizer;CF+Tri.,Trichoderma guizhouense NJAU 4742 (Tri.) applied with CF;OF,organic fertilizer;OF+Tri.,Tri.applied with OF;CK,no fertilizer;CK+Tri.,Tri.applied with CK.

4.Discussion

In this work,the optimized parameters of solid-state fermentation to produceT.guizhouense NJAU 4742 spores (7×109CFU g–1FW) were a material ratio of stevia residue to rice bran of 1:1,material thickness of 3 cm,initial moisture content of 60%,inoculum size of 10% and incubation time of 4 days.Similar to this study,the highest level ofClonostachys roseaspore production achieved was 3.4×109CFU g–1dry matter in solid-state fermentation using rice as raw material (Vicciniet al.2007).In this study,T.guizhouenseapplied with organic fertilizer significantly increased plant growth compared to organic fertilizer application alone,which is in agreement with previous reports (Qiuet al.2012;Caiet al.2015).In addition,the aboveground biomass ofT.guizhouenseapplied alone and in combination with chemical fertilizer was only slightly higher than those in CK and CF,respectively.This phenomenon may be attributed to mature compost representing optimal substrates for beneficial agents,thereby encouraging their establishment in the soil environment (Vinaleet al.2008).

Trichoderma guizhouenseapplication primarily reduced alpha richness (Chao1 index) and evenness (Shannon index) of fungal diversities in bulk soil,but had no influence on the rhizosphere.This finding is in line with the findings of Shenet al.(2015),who found that the alpha diversity of fungi was significantly decreased after two years of biofertilizer application.In contrast,Xionget al.(2017)found that the fungal richness and evenness were increased by aTrichoderma-amended bioorganic fertilizer treatment compared to an organic fertilizer treatment.In addition,Shade (2017) reported that there were nuanced relationships between alpha diversity and emergent properties of a microbial community.Our results suggest that the invasion ofT.guizhouensecan strongly affect the fungal community of bulk soil.

Microbial community analysis showed thatT.guizhouenseapplication primarily influenced bulk community structure compared to rhizosphere community structure.Many studies have reported that PGPF-containing fertilizers shape the bulk soil microbial community (Shenet al.2015;Xionget al.2017;Linet al.2020).In fact,the composition of the rhizosphere microbial communities is primarily induced by root exudates (Chaparroet al.2014).However,Bakkeret al.(2015) demonstrated that bulk microbial communities also had legacy effects of prior selections that influenced rhizosphere microbial community composition.Therefore,our next question was whether the affected bulk microbial communities provide a direct effect on rhizosphere microbial communities.

The results showed that the overlapping responsive OTUs induced byT.guizhouenseapplications were OTU_2,OTU_4,OTU_9 and OTU_11 in the bulk soil.Interestingly,three of the responsive OTUs in bulk soil(OTU_2,OTU_4 and OTU_9) were also stimulated in the rhizosphere soil.Moreover,all three OTUs belonged to the phylum Ascomycota,and OTU_2,OTU_4 and OTU_9 are classified asChaetomium,TrichodermaandChaetomiumat the genus level,respectively.These results reveal that some microbes could be induced byT.guizhouenseapplication under different practice strategies,in line with the application withTrichoderma-amended biofertilizer (Caiet al.2015),which increased the abundances of some potential taxa (including genusTrichoderma).Notably,the responsive OTU_3 (Phymatotrichopsis) that was stimulated in the rhizosphere soil byT.guizhouenseapplication was only induced in the bulk soil by the OF+Tri.treatment,suggesting that OTU_3 (Phymatotrichopsis) might be the key microbe in the OF+Tri.treatment which contributes to the promoting effect.Numerous studies have reported thatPhymatotrichopsisomnivorais a causal organism of cotton root rot (Mareket al.2009;Huet al.2011).However,to our knowledge,there is no report ofPhymatotrichopsisas a pathogen of corn crop.Interestingly,a nonpathogenicRalstoniasp.has resistance to another pathogenicRalstoniasp.(Weiet al.2015),suggesting that one genus can contain both good and bad members.Moreover,some enzyme activities ofTrichodermasp.were increased when the species interacted withPhymatotrichopsisomnivora(Guigón-Lópezet al.2015).Our results suggest that the interaction ofT.guizhouenseandPhymatotrichopsismight play an important role in promoting corn growth.

5.Conclusion

Stevia residue was successfully employed in solidstate fermentation for spore production ofTrichoderma guizhouenseNJAU 4742,and the maximum spore yield was up to 7×109CFU g–1FW.Trichoderma guizhouenseapplied with organic fertilizer generated the strongest soil productivity compared to either its application with chemical fertilizer or direct application.Interestingly,T.guizhouenseapplication under all the above-mentioned strategies could induce some common microbes.Nevertheless,some specific microbes (Phymatotrichopsis) were stimulated byT.guizhouenseapplied with organic fertilizer,possibly leading to high soil productivity.It is feasible to employ stevia residue for the eco-friendly fermentation ofT.guizhouense,which is strongly suggested for OF applications.

Acknowledgements

This research was supported by the National Key R&D Program of China (2018YFD0500201 and 2018YFD0201300),the Natural Science Foundation of Jiangsu Province,China (BK20180539),the National Science Foundation for Young Scientists of China(31801949),the National Science Foundation for Postdoctoral Scientists of China (2018M632318),the Agricultural Science and Technology Independent Innovation Fund Project of Jiangsu Province (CX(19)2026),and the Priority Academic Program Development of the Jiangsu Higher Education Institutions (PAPD).

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