Colonization by Klebsiella variicola FH-1 stimulates soybean growth and alleviates the stress of Sclerotinia sclerotiorum

ZHAl Qian-hang,PAN Ze-qun,ZHANG Cheng,YU Hui-lin,ZHANG Meng,GU Xue-hu,ZHANG Xiang-hui,PAN Hong-yu,ZHANG Hao#

1 College of Plant Protection,Jilin Agricultural University,Changchun 130118,P.R.China

2 College of Plant Sciences,Jilin University,Changchun 130062,P.R.China

Abstract

Sclerotinia stem rot,caused by Sclerotinia sclerotiorum,is a destructive soil-borne disease leading to huge yield loss.We previously reported that Klebsiella variicola FH-1 could degrade atrazine herbicides,and the vegetative growth of atrazine-sensitive crops (i.e.,soybean) was significantly increased in the FH-1-treated soil.Interestingly,we found that FH-1 could promote soybean growth and induce resistance to S.sclerotiorum.In our study,strain FH-1 could grow in a nitrogen-free environment,dissolve inorganic phosphorus and potassium,and produce indoleacetic acid and a siderophore.The results of pot experiments showed that K.variicola FH-1 promoted soybean plant development,substantially improving plant height,fresh weight,and root length,and induced resistance against S.sclerotiorum infection in soybean leaves.The area under the disease progression curve (AUDPC) for treatment with strain FH-1 was significantly lower than the control and was reduced by up to 42.2% within 48 h (P＜0.001).Moreover,strain FH-1 rcovered the activities of catalase,superoxide dismutase,peroxidase,phenylalanine ammonia lyase,and polyphenol oxidase,which are involved in plant protection,and reduced malondialdehyde accumulation in the leaves.The mechanism of induction of resistance appeared to be primarily resulted from the enhancement of transcript levels of PR10,PR12,AOS,CHS,and PDF1.2 genes.The colonization of FH-1 on soybean root,determined using CLSM and SEM,revealed that FH-1 colonized soybean root surfaces,root hairs,and exodermis to form biofilms.In summary,K.variicola FH-1 exhibited the biological control potential by inducing resistance in soybean against S.sclerotiorum infection,providing new suggestions for green prevention and control.

Keywords: sclerotinia stem rot of soybean,Klebsiella variicola FH-1,inducing resistance,root colonization,biofilm

1.lntroduction

Stem rot is caused bySclerotiniasclerotiorum,a fungal pathogen,which is one of the most destructive soil-borne plant pathogens present worldwide.It infects more than 500 types of plants globally,such as soybean,beans,sunflower,rape,carrot,and cabbage (Zhangetal.2022).Numerous studies have reported leaf lesions and seed reduction inS.sclerotiorum-infected soybean plants,which ultimately lead to the death of the infected plants within a few days of wilting (Zhangetal.2016).Moreover,the resistance ofS.sclerotiorumto fungicides presents a significant obstacle in effectively preventing and treating stem rot (Gossenetal.2001; Sierotzki and Scalliet 2013).Therefore,biological control,as a simple,environmentally friendly and effective method,can prevent and controlS.sclerotiorumand reduce its harm to soybean.Plant growth-promoting rhizobacteria (PGPR) play a crucial role in controlling plant defense against pathogens.They can change environmental conditions,and enhance plant growth and disease suppression (Van Weesetal.2008).Some microorganisms can promote plant growth through mechanisms,such as phytohormone production,soil nutrient solubilization,nitrogen fixation,and reduction of plant stress under different conditions (Jiangetal.2015).Plant growth-promoting bacteria can produce siderophores and different classes of antibiotics (Yangetal.2009).Some microorganisms protect plants against pathogens by regulating different defense enzymes of plants.These defense enzymes,including catalase (CAT),superoxide dismutase (SOD),peroxidase (POD),phenylalanine ammonia lyase (PAL),and polyphenol oxidase (PPO) play crucial roles in these processes (Van Oostenetal.2008).Application of rhizobacteria can induce resistance in plants against phytopathogens by enhancing the activities of genes that encode for enzymes involved in the production of secondary metabolites,pathogenesis-related (PR) protein,and certain regulatory proteins,which are involved in controlling other defense mechanisms (Selimetal.2017).Moreover,plants have other different important components in their immune system,such as plant defensins,that participate in plant defense against pathogens (Van Loonetal.1998; Lauetal.2020).

Effective colonization of plants by microorganisms in the plant rhizosphere is considered a prerequisite for promoting plant growth (Zhangetal.2011; Al-Alietal.2018).Stimulated by root exudates,microorganisms move to plant roots and rhizosphere soil in clusters and colonize the root surface by forming biofilms (Compantetal.2010; Budiharjoetal.2014; Cuietal.2019).Numerous benefits of biofilm-forming PGPR have been proposed in plants,such as facilitating plant growth,modulating phytohormone levels,and reducing biotic and abiotic plant stress (Van Loon and Bakker 2007; Lugtenberg and Kamilova 2009; Ahemad and Kibret 2014; Altaf and Ahmad 2017).Studies have shown thatBacillussubtilisforms a solid biofilm on the surface of tomato roots,thereby effectively controlling the pathogenRalstoniasolanacearum(Chenetal.2013).

Klebsiellahas been proven to be an excellent rhizosphere growth-promoting bacterium that can fix nitrogen and produce indoleacetic acid (IAA) and acid deaminase to promote plant growth (Iniguezetal.2004; Sachdevetal.2009; Singhetal.2015; Liuetal.2018).Furthermore,K.variicolais also used to control several plant diseases,such as tomato early blind,cabbage black spot,and tobacco red star disease (Lietal.2014).In addition,K.variicolaincreases the number of rhizobia in soil,which is beneficial for the optimization of soil environmental quality (Zhang Jetal.2021).Surprisingly,Zhang Metal.(2021) reported thatK.jilinsis2N3 degrades chlorimuron-ethyl in soil and promotes maize growth and induces resistance to northern corn leaf blight.This provides us with a new idea about the multifunctional application of microorganisms.

Finding a candidate biocontrol agent againstS.sclerotiorumwould be of great significance for the soybean industry.Our research group previously demonstrated that strain FH-1 has a positive effect on the remediation of atrazine-contaminated soil.The stem length,root length,and emergence rate of sensitive soybean crops planted in the remediated soil improved (Zhangetal.2020).In the present study,we evaluated the growth-promoting characteristics of the atrazinedegrading bacterium FH-1invitroand its promoting effect on soybean growth,and found that it could induce defense response toS.sclerotiorum.The effects of FH-1 on the activities of CAT,SOD,POD,phenylalanine ammonia-lyase,PPO,and MDA in soybean leaves underS.sclerotiorumstress were evaluated.The transcription levels of soybean defense-related genesPR10,PR12,AOS,CHS,PAL1,andPDF1.2were determined.In addition,the colonization of GFP-tagged FH-1 in soybean roots was monitored.Overall,the degrading bacteriumK.variicolaFH-1 can promote plant growth,induce resistance toS.sclerotioruminfection in soybean,and provide more experience basis for multifunctional application of microorganisms.

2.Materials and methods

2.1.Strains and seeds

Test strainsThe strainK.variicolaFH-1 from the China Typical Culture Collection (http://www.cctcc.org/; M2018334) was used in this study and was provided by the Department of Pesticides,Jilin Agricultural University,Jilin,China.The pathogenic fungusS.sclerotiorumwas provided by Dr.Pan Hongyu (College of Plant Science,Jilin University,Changchun,China).

Plant seedsSoybean seeds (Jidou 1) were provided by Dr.Pan Hongyu.Seeds with full grains were selected,sterilized with 75% ethanol,washed with disinfected distilled water,and further treated after air drying.

2.2.Microbial culture strains and pathogenic fungus

The strainK.variicolaFH-1 was cultivated on Luria-Bertani (LB) solid medium at 37°C for 12 h.A single colony was cultured in LB liquid medium at 37°C for 12 h at 150 r min–1,and the bacterial solution was centrifuged at 9 000 r min–1for 10 min.The bacterial cells were suspended in disinfected distilled water,and the concentration of the bacterial solution was adjusted to 1×108colony forming units (CFU) mL-1.The pathogenic fungusS.sclerotiorumwas maintained on potato dextrose agar solid medium for 24 h at 25°C in the dark.

2.3.Exploration of in vitro growth promotion of strain FH-1

Nitrogen fixation,phosphate solubilization,and potassium solubilizationFH-1 colonies were cultured on nitrogen-free Ashby medium (Purietal.2020) and Pikovskaya (PVK) medium and spot inoculated on potassium feldspar (PF) solid medium and incubated at 30°C for 5 days to observe the formation of hyaline zones around the bacterial colonies.The formation of clear transparent areas around the colonies is considered to indicate the ability of bacteria to dissolve phosphate.The nitrogen-free Ashby medium was used to qualitatively determine the nitrogen-fixing capacity of FH-1,while the PVK medium was used to qualitatively determine the phosphate solubilization activity of FH-1 (Nautiyal 1999).The PF solid medium was used to assess the potassium solubilization capacity of FH-1 (Cuietal.2019).

Siderophore productionSiderophore production by FH-1 was determined qualitatively using the universal Chromeazurol S (CAS) agar plate assay.The CAS agar medium consisted of 1 mmol L–1CAS,10 mL FeCl3·6H2O made in 10 mmol L–1HCl,and 2 mmol L–1N,N-hexadecyltrimethylammonium bromide.The reagent was autoclaved separately and added to 500 mL of LB medium (Tanketal.2012).Freshly grown FH-1 cultures were spot inoculated onto the CAS agar medium and incubated at 30°C for 3 days.Orange halos formed around the colonies indicated siderophore biosynthesis (Yuetal.2011).

lAAquantificationFor inoculum standardization,strains were initially transferred to nutrient broth for 48 h and incubated at 30°C.The optical density (OD) was then adjusted to 1.0 (107–108CFU mL-1).Afterward,the strains were transferred (5%,v/v) to nutrient broth containing tryptophan (100 μg mL-1) and incubated at 30°C in the dark for 72 h (Benetal.2018).Culture supernatants were recovered after centrifugation at 12 000 r min–1for 5 min.Auxin production was determined by mixing 3 mL of the supernatant with 3 mL of Salkowski’s reagent (1.875 g of FeCl3·6H2O,100 mL of H2O,and 150 mL of H2SO4).The mixture was incubated at 30°C for 15 min in the dark.Phytohormone production was determined by measuring absorbance at 530 nm with a spectrophotometer,and the phytohormone level was calculated from a standard curve constructed with pure IAA (Penrose and Glick 2003).

2.4.Growth promotion assay

Preparation of soybean seedlings and plant treatmentFirst,intact seeds were selected,surface bacteria were removed with 75% ethanol,and the ethanol on the seed surface was washed with sterile water.Soybean seeds (3 seeds per pot) were sown in grass charcoal soil/vermiculite (1:1) pots (pot diameter×height: 45 cm×45 cm).The temperature of the culture room was maintained at 26°C with sunlight for 12 h per day on average and 35% relative humidity.Water was applied occasionally to keep the soil sufficiently moist.Soybean plants were grown under these conditions until the late V2 stage (having at least 2 fully expanded leaves).We ensured no significant difference in the growth of the 3 plants per pot.Soil moisture was maintained with sterile water,and the experiment was repeated 3 times.

Detection of soybean growth characteristicsThe growth promotion effect of 15 mL FH-1 (108CFU mL-1) on the V2 stage plants was measured at 7 and 14 days after bacterial inoculation.Plant height,fresh weight,dry weight,and root growth were measured in the treatment and control groups (Attiaetal.2020).In addition,each root sample was cleaned with sterile distilled water until no soil was left on the root surface.The samples were scanned using an EPSON V330 Scanner (Epson America Inc.,Long Beach,CA,USA),and root morphological characteristics,such as total length,total surface area,total volume,root tip number,branch number,and hybridization number,were analyzed using WinRyS (Zhang Metal.2021).

Determination of root activityThe effect of 15 mL FH-1 (108CFU mL-1) on the root activity of V2 stage plants was measured at 12,24,48,72,96,and 120 h after bacterial inoculation.The triphenyltetrazolium chloride (TTC) method was used to determine root vigor (Comasetal.2000).Soybean root tips weighing 0.5 g were placed in a test tube containing 5 mL of 0.4% TTC and 5 mL phosphate buffer (pH 7.5).The roots were completely immersed in the reaction solution,and the reaction was carried out in the dark in a 37°C water bath for 1 h.The reaction was terminated by adding 2 mL of concentrated sulfuric acid.For the blank test,sulfuric acid was added first,followed by the root sample,and then,the other steps were performed as mentioned above.After the reaction was terminated,the roots were removed,blotted on a filter paper,and added to the mortar.Then,5 mL of acetone and a little quartz sand were added to the mortar,and the roots were ground to obtain the red-colored triphenylformazan (TTF) extract.The liquid phase was transferred to a 25-mL volumetric flask and fixed with acetone.Absorbance was measured at 485 nm by using a 1-cm optical diameter cuvette,and the standard curve was generated to determine TTF content according to the measured absorbance value (Jiangetal.2013).The root vigor of soybean was then calculated on the basis of TTF content.

2.5.Biological control analysis

Bacterial induction and sickness infectionSoybean growing to the V2 leaf stage was selected as the experimental object.Then 15 mL of the FH-1 cell suspension (108CFU mL-1) was inoculated into the soil where the plants were grown for 12 h before the infection ofS.sclerotiorum.The plugs withS.sclerotiorumwere placed side down in the center position on each leaf.All treated and control plants were then misted with water and covered with plastic bags for 2 consecutive days to increase relative humidity.Leaf samples were harvested at 12,24,48,72,96,and 120 h after inoculation,immediately immersed in liquid nitrogen,and kept at -80°C until they were subject to extraction for enzyme and RNA analyses.The following treatments were considered: (1) Soybean plants without any treatment were referred to as the healthy control (-FH-1-S); (2) soybean plants infected withS.sclerotiorumwere referred to as the infected control (-FH-1+S); (3) soybean plants treated withK.variicolaFH-1 (+FH-1-S); and (4) plants treated withK.variicolaFH-1 and then infected withS.sclerotiorum(+FH-1+S).

Evaluation of disease severityEighteen plants from the (+FH-1+S) and (-FH-1+S) experimental units were visually rated for disease at 96 h.The leaves were also photographed to obtain digital images.From the images,the disease spot diameter was calculated using the Lesion Assay Software ImageJ (National Institutes of Health) (Azabouetal.2020).The disease parameters AUDPC in 96 h are used for disease quantification.AUDPC is calculated using the mean of all plants in (+FH-1+S) and (-FH-1+S) experimental units.The AUDPC are calculated as follows:

whereyi=disease spot diameter at dayi,ti+1–ti=day interval between two ratings,n=number of ratings (Chungetal.2011).

Measurement of defense enzyme activityLeaf samples were collected at 12,24,48,72,96,and 120 h for enzyme activity analysis.Three samples were collected from each treatment group: (-FH-1-S),(-FH-1+S),and (+FH-1-S) and (+FH-1+S).POD activity was assayed using guaiacol as a substrate at 470 nm.The reaction mixture contained 50 mmol L-1potassium phosphate buffer (pH 6.4),0.3% (w/v) guaiacol,0.3% (w/v) H2O2,and 100 μL of crude enzyme extract in a final volume of 300 μL.POD activity was detected by monitoring the formation of tetraguaiacol and the consequent increase in absorbance at 470 nm (Senthilrajaetal.2013).To assess PAL activity,0.5 g of blade was extracted with 50 mmol L–1H3BO3buffer (pH 8.8) containing 8 mmol L–1β-mercaptoethanol (HSCH2CH2OH) and 1% polyvinylpyrrolidone.The homogenate was centrifuged at 12 000 r min–1for 15 min.PAL activity was measured at 290 nm by using L-phenylalanine as the reactant (Einhardtetal.2022).SOD activity was evaluated based on the nitro blue tetrazolium reduction method (Jogaiahetal.2020).CAT activity was measured according to Pateletal.(2017).The reaction mixture consisted of 1.5 mL of 100 mmol L–1potassium phosphate buffer (pH 7),0.5 mL of 7.5 mmol L–1hydrogen peroxide,and 75 μL enzyme.The sample volume was adjusted to 3 mL by adding distilled water.A decrease in sample absorbance was recorded at 240 nm for 1 min by using a spectrophotometer.PPO activity was determined using catechol as a substrate.Formation of the yellow color product benzoquinone after catechol oxidation was measured at 495 nm (Mayeretal.1966).To determine MDA content in soybean leaves,the leaves (0.5 g) were ground evenly in 5 mL of 5% trichloroacetic acid.Then,the ground sample was centrifuged at 12 000 r min–1at 4°C for 15 min.The supernatant was added to 2-thiobarbituric acid in a test tube.The mixture was then heated in a water bath at 98°C for 10 min.After the sample was cooled to room temperature,the absorbance of the mixture was measured at 532,600,and 450 nm (Du and Bramlage 1992).MDA content was calculated using the following formula: cMDA (mmol g-1)=6.45×(OD532-OD600)-0.56×OD450.

Defense-related gene expression assayQuantitative real-time PCR was used to analyze the expression levels of defense-related genes in soybean plants inoculated with only sterile water (control),S.sclerotiorumor FH-1,and bothS.sclerotiorumand FH-1.The leaves were sampled at 0,12,24,48,72,and 96 h after inoculation withS.sclerotiorum.The samples were frozen in liquid nitrogen and placed in a refrigerator at -80°C for later use.The expression levels were determined using a Roche LightCycler?480 (Roche Diagnostics Corporation,Indianapolis,IN,USA).Total RNA was extracted and purified using the TRIzol Up Plus RNA Kit (TransGen Biotech,Beijing,China).Total RNA for qPCR was extracted and purified using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech),and cDNA was synthesized from 1 μg RNA according to the manufacturer’s instructions.Transcriptspecific primers were verified through PCR.Realtime PCR was performed using the PerfectStart Green qPCR SuperMix (TransGen Biotech),with 3 biological replicates per run,including the reference gene.The reaction system contained 5 μL Eva Green Master Mix,2 μL ultrapure water,1 μL (10 μmol L-1) primers,and 2 μL cDNA (100 ng μL-1dilution).The amplification conditions were 95°C for 2 min to activate the heat-activated recombinantTaqDNA polymerase,followed by 34 cycles of amplification at 95°C for 15 s,60°C for 20 s,72°C for 15 s,and 95°C for 15 s.Each experiment was repeated 3 times.Quantitative RT-PCR data were analyzed using the comparative cycling threshold (Ct) method,presenting a 2–ΔΔCtfold change in gene expression (Livak and Schmittgen 2001).

The expression levels of 6 defense-related genes (PDF1.2,PAL1,AOS,PR12,PR10,andCHS) that encoded for enzymes involved in the production of secondary metabolites,PRprotein,and plant defensins were determined (Hayashietal.2008; Upchurch and Ramirez 2010).The primers used in this experiment are listed in Appendix A.

2.6.Bacterial colonization assay

Formation of FH-1 biofilmStrain FH-1 was grown in LB at 37°C for 12 h.The bacterial cells were collected and washed 2 times with sterile water.The bacterial density was adjusted to 1×108CFU mL-1.Then,100 μL of the bacterial suspension was inoculated into 24-hole polystyrene plate wells filled with 1 mL of fresh LB (Jainetal.2013),and sterile LB was used as a control.After incubation at 25°C (12,24,48,72,96,and 120 h),the biofilm formed was dyed with crystal violet aqueous solution (0.1%) for 20 min,washed 3 times with sterile distilled water,and immediately decolorized with 1 mL of 95% ethanol for 20 min.Then,1 mL of the solution was transferred to a cuvette,and the absorbance of crystal violet was measured at OD570(Peetersetal.2008),which was used to evaluate biofilm formation by FH-1.Moreover,a 1 cm×1 cm sterile cover glass was placed in a 6-hole polystyrene plate well containing 2 mL fresh LB medium.Then,20 μL (1×108CFU mL-1) FH-1 bacterial suspension was added to the LB medium and dyed with crystal violet.The unbound dye solution on the cover glass was washed off again,air-dried,and magnified 10×40 times under an optical microscope to observe the biofilm formed on the cover glass at different times.Each treatment was repeated in triplicate,and the test was performed in duplicate.

Construction of GFP-labeled strain FH-1The pgfp48 plasmid containing the green fluorescent protein gene was introduced into the wild-type FH-1 strain through electroporation.Bacteria were cultured in LB broth medium incubated at 30°C for 12 h.Then,1% bacterial suspension was inoculated into fresh 100 mL LB liquid medium and cultured on a shaker at 28°C until OD600reached 0.5 (Zhuetal.2021).The cells were then centrifuged at 4°C and 8 000 r min–1for 15 min.After washing the cells 3 times with precooled 10% glycerol,the supernatant was discarded and the cells were resuspended in 1 mL of 10% glycerol to prepare competent cells.Then,100 μL of competent FH-1 cells was mixed with 5 μL (50 ng) of the pgfp48 plasmid,and the mixture was subjected to shock in an electric shock transformation cup (2 mm).The transformed cells were immediately added to 700 μL SOC medium,resuscitated at 37°C for 2 h,and evenly coated on an LB plate containing 50 μg mL-1kanamycin.A transformant stably emitting green fluorescence under a fluorescence microscope was obtained and termed FH-1-gfp (Yi and Kuipers 2017).

Fluorescence microscopy of FH-1-gfp colonization of soybean rootsSoybean seeds were surface sterilized and germinated.The germinated seedlings were transplanted into autoclaved soil and grown at 25°C under a 12-h photoperiod.When the seedlings reached the V2 stage,their roots were immersed for 6 h in the bacterial suspension containing 108CFU mL-1FH-1-gfp bacteria,and then inoculated and replanted into the soil.Control test tube seedlings were treated with sterile saline in the same manner.Two days after replantation,the roots were removed from the soil,and the surface soil was washed away.The roots were then cut into 1-cm pieces,preserved with 90% glycerin,and observed through CLSM (FV1000,Jilin University College of Plant Sciences,Changchun,China) at an excitation wavelength of 488 nm to determine FH-1-gfp colonization of tissues.Images were obtained using Olympus FV10-ASW 4.0 Software (Posadaetal.2018).

Scanning electron microscopy of FH-1 coloniza- tionThe colonization ability of FH-1 on soybean roots was observed through scanning electron microscopy (SEM).The roots were rinsed 3 times with sterile water.Then,the roots were immersed in 2.5% (v/v)glutaraldehyde at 4°C for 6 h and washed 3 times with phosphate buffer (Pathanetal.2010).The root samples were dehydrated in 50,70,80,90,and 100% ethanol for 15 min each and dried using a vacuum freeze dryer FD-1A-50 (Tianjin Bilang Experimental Instrument Manufacturing Co.,Ltd.,Changchun,China).The dehydrated soybean roots were plated by ion sputtering (Quorum,SC76220,Sputter Coatinger) and observed through SEM (Zeiss,GeminiSEM 450,Shanghai Laser Spectrum Instrument Analysis Technology Co.,Ltd.,Changchun,China) (Yaoetal.2016).

2.7.Statistical analysis

All experimental data were analyzed using Origin 2019.Analysis of variance was performed to determine the significance of observed differences by using the SPSS Software Package (SPSS 19.0 for Windows,USA).GraphPad Prism Program’st-tests and one-way ANOVA were used for significant differences analysis.The bars indicate standard deviation (SD) of mean.Each experiment had 3 replicates.Asterisks indicate significant differences (＊,P＜0.05;＊＊,P＜0.01;＊＊＊,P＜0.001,＊＊＊＊,P＜0.0001; NS,no statistical difference).

3.Results

3.1.Plant growth-promoting activity of the bacterial strain FH-1 in vitro

The diameter of the halo around the bacterial colony was used to evaluate potential of the strain FH-1 as promoting plant growth bacteria.The strain generated a noticeable halo of disintegration on the nitrogen-free Ashby medium after 3 days of incubation at 30°C (Fig.1-A).Halos of disintegration were also observed on PVK and PF media (Fig.1-B and C).These results indicate that strain FH-1 can effectively fix nitrogen and dissolve phosphorus and potassium.On CAS detection medium,strain FH-1 produced a noticeable orange halo (Fig.1-D),demonstrating its excellent ability and high potential to produce siderophores.Moreover,the red hue in the FH-1 culture supernatant containing L-tryptophan was significantly deeper than that in the culture supernatant without L-tryptophan,which indicated that FH-1 can synthesize IAA by using L-tryptophan as a precursor (Fig.1-E).According to the IAA standard curve,the IAA concentration produced by FH-1 was (35.216±0.34) mg mL-1after 72 h,and the IAA content in culture media without L-tryptophan was (1.13±0.32) mg mL-1.

Fig.1 Exploration of in vitro growth promotion of strain FH-1.A,nitrogen fixation activity of strain FH-1.B,phosphate-solubilizing activity of strain FH-1.C,potassium dissolving activity of strain FH-1.D,siderophore production activity of strain FH-1.E,the indole-3-acetic acid (IAA) producing activities of strain FH-1 were –L-tryptophan and +L-tryptophan,respectively.

3.2.Promotion of biomass accumulation

Compared with the control group,the plant height of soybean increased at 7 and 14 days after FH-1 treatment (Fig.2-A and B).Plant height,fresh weight,and dry weight of aboveground portions increased by 47.90,26.40,and 16.18%,respectively,at 7 days after FH-1 treatment and by 54.16,26.61,and 14.71%,respectively,at 14 days after FH-1 treatment (Fig.2-C–E).

Fig.2 Effect of strain FH-1 on soybean growth and development.A and B,promoting effect of strain FH-1 on soybean growth in pot experiment.Plant height,dry weight and fresh weight were measured 7 and 14 d after inoculation with strain FH-1.C,effect of soybean plant height by strain FH-1.D,effect of soybean dry weight by strain FH-1.E,effect of soybean fresh weight by strain FH-1.GraphPad Prism Program’s t-test was used for significant differences analysis.The bars indicate standard deviation (SD) of mean (n=3).Asterisks indicate significant differences (＊,P＜0.05; ＊＊,P＜0.01; ＊＊＊,P＜0.001; NS,no statistical difference).

3.3.Root development in soybean

Root scanning analysis (Fig.3-A and B) showed that strain FH-1 improved soybean root development to different degrees on 7 and 14 days.The total length,surface area,and volume of roots increased by 198.70,189.90,and 106.10%,respectively,at 7 days and by 77.52,46.40,and 24.99%,respectively,at 14 days (Fig.4-A–C).Compared with the control group,the total number of root tips and the number of branches increased 2.6- and 4-fold,respectively,at 7 days and 1.75- and 2.18-fold at 14 days,respectively,in the treatment group (Fig.4-D and E).TTC is reduced to generate TTF,which is red and water insoluble.The generated TTF is relatively stable and is not automatically oxidized by oxygen in the air.Therefore,TTF can be used as an index to measure root activity.After adding strain FH-1 for 12 h,the root activity of soybean increased sharply,which was 105.0 and 95.4% higher than that in the control group at 12 and 24 h,respectively.The effect gradually weakened with time (Fig.4-F).These results indicated that FH-1 could improve the growth and development of the soybean root system.

Fig.3 Effects of strain FH-1 on morphological characteristics of soybean roots after 7 days (A) and 14 days (B).

Fig.4 Effect of strain FH-1 inoculation on soybean.A,root length.B,total root surface.C,total root volume.D,number of root tips.E,number of root branch.F,root activity.GraphPad Prism Program’s t-test was used for significant differences analysis.The bars indicate standard deviation (SD) of mean (n=3).Each experiment had three replicates.Asterisks indicate significant differences (＊,P＜0.05; ＊＊,P＜0.01; NS,no statistical difference).

3.4.Biocontrol efficacy

The protective test showed that after inoculation ofS.sclerotiorumon soybean leaves for 24 h,obvious water-soaked chlorosis spots began to appear in the control group,while the area of chlorosis spots on soybean leaves pretreated with FH-1 was smaller than that in the control group.In addition,the therapeutic effect of the FH-1 treatment group gradually weakened with time.Surprisingly,after 96 h,in the control group,the soybean leaves completely turned yellow,whereas healthy areas were still observed on the soybean leaves pretreated with FH-1 (Fig.5-A).Then,in the area under the disease progression curve of soybean infected withS.sclerotiorum,the (+FH-1+S) treatment effect was highly significant (P＜0.001) (Fig.5-B).The results showed that soybean leaves were infected withS.sclerotiorum(AUDPC value range) after spraying in 96 h: (–FH-1+S) treatment was (10.85 to 213.38) and (+FH-1+S) treatment was (6.15 to 147.33).Moreover,disease control with strain FH-1 was also observed in Fig.5-B,with a reduction of 42.2% of the AUDPC in soybean inoculated withS.sclerotiorumin 48 h.The experiment showed that compared withS.sclerotiorumtreatment alone,strain FH-1 pretreatment was significantly more efficient at reducing lesion progress of lesion formation on soybean leaves caused byS.sclerotioruminfection and had an obvious significant positive effect on the resistance of soybean againstS.sclerotiorum.

Fig.5 Inducing resistance in soybean against Sclerotinia sclerotiorum infection by strain FH-1.Appearance of soybeans leaves challenged with S.sclerotiorum in absence/presence of K.varicola FH-1.A,12,24,48,72 and 96 h following inoculation.B,area under the disease progress curve (AUDPC) in soybean leaves with S.sclerotiorum treated (12–96 h) with strain FH-1 and sterile water.GraphPad Prism Program’s t-test was used for significant differences analysis.The bars indicate standard deviation (SD) of mean (n=3).Asterisks indicate significant differences (＊＊＊,P＜0.001).

3.5.Plant defense enzyme activity

To study whether FH-1 can enhance the defense response activity of soybean cells,the activities of defense-related enzymes in soybean plants were investigated.The results showed that,under the effect ofS.sclerotiorum,POD accumulation in soybean leaves pretreated with FH-1 began to continue to increase from 48 h and reached its peak at 72 h.Compared with the (-FH-1+S) treatment group,it increased by 35.7% (Fig.6-A).The accumulation of PPO and CAT in the (+FH-1+S) treatment group was higher than that in the (-FH-1+S) treatment group within 120 h and reached the peak at 72 h,which increased by 72.2 and 43.5%,respectively,compared with that in the (-FH-1+S) treatment group (Fig.6-B and C).Compared with the (-FH-1+S) treatment group,PAL activity in the (+FH-1+S) treatment group increased by 19.0 and 16.4% at 24 and 48 h,respectively (Fig.6-D).At 48 h after pathogen inoculation,SOD activity was observed at higher levels in FH-1-pretreated soybean leaves,which was 44.0% higher than that of in the (-FH-1+S) treatment group (Fig.6-E).Furthermore,MDA activity in the (+FH-1+S) treatment group was 44.2–67.0% lower than that in the (-FH-1+S) treatment group from 24 to 96 h,which indicated that FH-1 effectively prevented the damage caused by S.sclerotiorum to soybean leaf tissue (Fig.6-F).

Fig.6 Effects of strain FH-1 on the activities of defense enzymes in leaves of soybean.A,peroxidase (POD).B,polyphenol oxidase (PPO).C,catalase (CAT).D,phenylalanineammonialyase (PAL).E,superoxide dismutase (SOD).F,malondialdehyde (MDA).SPSS Program’s one-way ANOVA was used for significant differences analysis.The bars indicate standard deviation (SD) of mean.Capital letters (A,B,C,D) on bars indicate significant difference between different treatments at the same time based on Duncan’s significant differences test (P＜0.01).Lowercase letters (a,b,c,d) on bars indicate significant difference between the same treatment at different times based on Duncan’s significant differences test (P＜0.05).

3.6.Expression of defense-related genes in soybean

To determine whether FH-1 can activate the expression of defense-related genes in soybean,the expression levels of 6 genes in soybean plants subjected to different treatments were tested.The average schedule of defense-related genes in leaves of soybean infected withS.sclerotiorumat 12,24,48,72,and 96 h is shown in Fig.7.Six genes are possibly related to soybean disease resistance,namelyPR10,PR12,PDF1.2,CHS,AOS,andPAL1.The expression profiles of all 6 genes inS.sclerotioruminoculated leaves showed similar patterns.Compared with the (-FH-1+S) treatment group,PR10expression in the FH-1-pretreated soybean leaves was increased by 29.5-fold at 24 h after inoculation withS.sclerotiorum.PR12expression in the (+FH-1+S) treatment group was also significantly increased at 24 h and was 19.4-fold higher than that in the (-FH-1+S) treatment group.At the earliest,that is,24 h after (+FH-1+S) treatment,PDF1.2expression in soybean leaves reached the highest level,much higher than that in the (-FH-1+S) treatment group.Moreover,CHSexpression in the (+FH-1+S) treatment group was always higher than that in the (-FH-1+S) treatment group within 96 h and reached the peak at 24 h.AOSexpression in the (+FH-1+S) treatment group was delayed at 12 h,and after 24 h,the expression level was significantly upregulated and continuously higher than that in the (-FH-1+S) treatment group.However,the expression trend ofPAL1was low.The expression was only upregulated by 4.8-fold in 24 h,and then showed no obvious upregulation.These results indicate thatK.variicolaFH-1 can enhance soybean resistance toS.sclerotiorumby activating signaling pathways related to synthesis of secondary metabolites,PRprotein,and plant defensins.

Fig.7 The expression levels of 6 defense-related genes of secondary metabolites enzymes,pathogenesis related (PR)-protein and plant defensins in soybean leaves (PR10,PR12,PDF1.2,CHS,AOS,and PAL1).Three samples were collected from each treatment including (-FH-1-S),(-FH-1+S),(+FH-1-S),and (+FH-1+S).Using soybean Actin gene as internal standard,the expression value of single gene was standardized.SPSS Program’s one-way ANOVA was used for significant differences analysis.The bars indicate standard deviation (SD) of mean (n=3).Lowercase letters (a,b,c,d) on bars indicate significant difference between the same treatment at different times based on Duncan’s significant differences test (P＜0.05).

3.7.Biofilm formation

The ability of FH-1 to form biofilms was determined using the crystal violet chromogenic method.The morphology of the biofilm formed by FH-1 and stained with crystal violet at different time periods was observed under a microscope (Fig.8-A–F).The results showed that FH-1 bacteria adhered less before 24 h,and most of them were scattered.After 48 h,some dispersed bacteria connected with each other,while some overlapped and bonded into clusters,forming a typical biofilm structure.The number of bacteria that adhered to the cover glass increased significantly.An ultraviolet spectrophotometer was used to quantify the biofilm formation of strain FH-1 at OD570(Fig.8-G).Significant difference in biofilm formation was observed with time; biofilm formation reached the maximum at 96 h.In the later stage,biofilm formation tended to stabilize.

Fig.8 Detection of biofilm formation ability of strain FH-1.A–F,the formation of strain FH-1 at 12,24,48,72,96 and 120 h,respectively (observed by optical microscope).Scale bar=100 μm.G,the biofilm formed by strain FH-1 at different time periods was stained by crystal violet and quantified by ultraviolet spectrophotometer OD570.SPSS Program’s one-way ANOVA was used for significant differences analysis.The bars indicate standard deviation (SD) of mean (n=3).

3.8.Colonization of soybean roots by strain FH-1

To detect whether FH-1 can colonize the soybean root system,the pgfp48 plasmid was transferred to FH-1 through electroporation,and the GFP-labeled strain FH-1-gfp with green fluorescence was constructed.Its growth rate was similar to that of the wild type,and their phenotypes were also the same.This indicates that the introduced fluorescent plasmid does not interfere with the growth and metabolism of normal strains (these data are not displayed).CLSM was used to observe soybean roots inoculated with FH-1-gfp and the blank control.No green fluorescence was observed on the surface of soybean roots inoculated without FH-1-gfp (Fig.9-A and C).After inoculation with FH-1-gfp,as observed under CLSM,many GFP-labeled bacterial cells adhered along the root hairs and colonized,and a small number of cells lived in intercellular spaces (Fig.9-B).Furthermore,the green fluorescent-labeled strains aggregated on the root surface in a dense settlement mode (Fig.9-D).

Based on the SEM images (Fig.10-A and B),no bacterial colonization was observed on the root surface of soybean plants in the control group.In the treatment group,FH-1 could well colonize soybean roots and mainly adhered along the root surface forming biofilm (Fig.10-C and D).The results showed that strain FH-1 had a strong colonization ability,and the colonization site in the root system was consistent with that observed through CLSM (Fig.9).

Fig.10 SEM images of strain FH-1 colonizing on the soybean roots.A and B,control of sterile water inoculation.C and D,the strain FH-1 emerged on the root surfaces and formed dense microcolony on the surface of root.

4.Discussion

4.1.The influence of strain FH-1 on the growth of soybean seedlings

This study provides new evidence for the growthpromoting effect ofK.variicolaFH-1 on soybean and its biological control application.Theinvitroexperiment indicated that strain FH-1 is an excellent growth-promoting bacterium that could fix nitrogen,dissolve phosphorus and potassium,and produce a siderophore and IAA (Fig.1).Furthermore,compared with the uninoculated control,FH-1 could promote the length of soybean roots and aerial parts and the number of roots (Figs.2–4).Some previous study showed that microorganisms can use siderophores to inhibit the growth of pathogenic microorganisms and induce resistance to plant diseases,and they may also favor plant nutrient acquisition,acting as a direct mechanism in promoting plant growth (Ramamoorthyetal.2001; Martinez-Viverosetal.2010).IAA,as one of the most effective phytohormones controlling plant development,is commonly produced by plant rhizosphere microorganisms and can regulate the growth and development of the plant root and aerial part (Etchellsetal.2016; Karthikaetal.2020; Zhouetal.2021).These results suggest that FH-1 could promote the growth of soybean seedlings.

4.2.Strain FH-1 induces soybean defense enzyme activity against S.sclerotiorum infection

The pot experiment showed that strain FH-1 could induce soybean to resistS.sclerotiorum.As expected,the average infection rates calculated by AUDPC were significantly higher for untreated soybean having a faster disease development compared to the soybean that was treated with strain FH-1.Here,we found that afterS.sclerotioruminfection,the activities of PPO,POD,SOD,CAT,and PAL in FH-1-treated soybean leaves were improved at different time points and to different degrees compared with the control (Fig.6).Rhizosphere bacteria enhance the resistance of plants to pathogenic bacteria,which is usually combined with the involvement of cell defense response activities,such as rapid accumulation of hydrogen peroxide and increase in defense-related enzyme activities (Van Weesetal.2008; Jiangetal.2018).PPO catalyzes the conversion of phenols into quinones,thus directly participating in disease resistance and killing or inhibiting pathogenic bacteria (Lietal.2016).POD as a class of PR protein is stimulated by pathogenic infection in host plant tissues and limits the spread of the pathogen by creating structural barriers (Wangetal.2009; Kumaretal.2016).SOD and CAT are plant cell defense enzymes that remove excessive reactive oxygen species and effectively prevent the invasion of the pathogen (Zhang Metal.2021).PAL is involved in the biosynthesis of active metabolites,lignin,and other phytochemicals that restrict the further growth of pathogens around the infected area (Shenetal.2014).These defenserelated enzymes in plants are activated when pathogen invasion induces plant systemic resistance (Van Loon and Bakker 2005,2007).Moreover,normal plant growth cells are constantly regulated by the active antioxidant enzyme system,which may lead to plant cell damage under pathogen and abiotic stresses,producing MDA and hence indicating cell death or decay (Liuetal.2021).In the current study,the MDA content of theS.sclerotiorum-challenged soybean was significantly higher than that of the FH-1-treated soybean.This indicates that plants encountered higher cellular damages due to pathogenic infection.However,the FH-1-treated soybean resisted the infection,maintaining cellular stability and producing lesser MDA content.Results of the enzyme activity test confirmed that strain FH-1 can activate soybean defense-related enzyme activities,thus limitingS.sclerotioruminfection.

4.3.Strain FH-1 induces the expression of soybean defense genes against S.sclerotiorum infection

In the presence of pathogens,we observed that the resistance-related genesPR10,PR12,PDF1.2,AOS,andCHSwere highly expressed in FH-1-treated soybean leaves,and they showed similar expression patterns (Fig.7).Many PR protein genes are upregulated in the process of plant resistance and trigger a wide range of protective responses in plants at a high level,thus inhibiting or reducing the colonization and development of pathogens.In particular,the expression levels ofPR10andPR12were significantly upregulated in the (+FH-1+S) treatment group compared with the (-FH-1+S) treatment group.The PR12 protein,as a defensin protein,can cause plasmolysis of fungal cells,thereby inhibiting the infection of pathogenic fungi.When pathogens invade,the PR10 protein mainly accumulates around the damaged parts of plants and is induced by other environmental stresses; it participates in the defense mechanism of plants (Jiangetal.2015; Patiletal.2021).In response to the attack ofS.sclerotiorum,FH-1-induced defense protein provided significant protection to soybean through the defense mechanism.PAL1encodes for enzymes of the phenylpropanoid pathway.Several studies have shown thatPAL1is expressed in response to various environmental stimuli,including pathogenic infection by fungi and bacteria,and other biotic stressors.Some genes related to the phenylpropanoid pathway are involved in defense during the interaction between soybean and other pathogens (e.g.,RhizoctoniasolaniandPhytophthorasojae) (Chenetal.2009; Xuetal.2012).Our results showed that in the presence of pathogens,strain FH-1 increasedPAL1expression from 24 h,but it was not significantly upregulated,which was similar to the result of PAL enzyme activity determined (Bazzaloetal.1985; Callaetal.2009; Zhaoetal.2009).This may be related to the mode of action of inducing expression (Jungetal.2011).CHS,as one of the major enzymes of the flavonoid pathway,is involved in plant resistance to diseases,especially in soybean defense response toS.sclerotioruminfection (Zhaoetal.2009).The (+FH-1+S) treatment increasedCHSexpression,and higher content of flavonoids is helpful to slow down the progress ofS.sclerotioruminfection in soybean (Chenetal.2009; Xuetal.2012).CHScan also improve nodulation and the nitrogen utilization efficiency by allowing the direct interaction betweenRhizobiumpresent in soybean soil andK.variicolaFH-1 and promote the growth and development of soybean plants (Hirschi 2004).

Plant defensin (encoded byPDF1.2) enables the defense response of plants to adverse stresses such as fungal invasion,virus infection,and environment.Propylene oxide synthase (encoded byAOS) is the key enzyme in jasmonic acid (JA) synthesis and plays a crucial role in the defense system of plants.These two genes are closely related to the JA pathway (Samainetal.2019).After pretreatment with strain FH-1,PDF1.2expression in soybean leaves inoculated withS.sclerotiorumincreased rapidly at 12 h,whereasAOSexpression was delayed,but it increased significantly after 24 h.The overall trend however was higher than that in soybean leaves inoculated withS.sclerotiorumalone (Zhaoetal.2007).This suggested that JA- and ET-dependent pathways were induced byS.sclerotioruminfection in soybean.However,in addition to the JA/ET-mediated defense response,the salicylic acid (SA)-mediated defense response is involved in defense againstS.sclerotiorum(Guo and Stotz 2007).In another study,PDF1.2was locally induced by compounds that produce AOS in the plant tissue so as to directly participate in defense regulation or its reaction pathway (Mitteretal.1998).Our results showed that,multiple phytohormoneresponsive defense genes such asCHS,PDF1.2,AOS,PR10,andPR12were significantly induced in the resistant genotype,suggesting the coordinated activation between JA/ET- and SA-mediated signal pathways in FH-1-induced soybean resistance toS.sclerotiorum.

4.4.Strain FH-1 colonizes soybean roots in the form of biofilm

Biofilm,as a complex multi-cell community,can effectively help bacteria colonize the root surface of host plants (Beauregardetal.2013; Vlamakisetal.2013).By forming these biofilms,rhizosphere microorganisms realize their ability to and induce plant growth and development (Yangetal.2018).The lasting and stable colonization of bacteria in roots is considered a vital factor for promoting plant growth (Caoetal.2011; Zhaoetal.2011; Chowdhuryetal.2013; Maetal.2018).In this study,the beneficial strain FH-1 was labeled with a green fluorescent protein and was visualized through CLSM during rhizosphere colonization of soybean seedlings.Colonization of the soybean root surface,root hair,and root depression occurs in the form of cell aggregates or microcolonies (Fig.9).SEM displayed that FH-1 infiltrated into the soil to form aggregates and adhere to the root surface (Fig.10).Plant root exudates can induce bacterial chemotaxis to the surface of plant roots,allowing these bacteria to thus colonize the plant roots (Tanetal.2013; Zhangetal.2014; Cuietal.2019).Some components in soybean rhizosphere exudates may attract strain FH-1 to colonize the roots by inducing chemotaxis and release some substances to induce a soybean defense response againstS.sclerotioruminfection.

5.Conclusion

As an environmental-friendly and sustainable method,use of biological control agents can reduce pathogenmediated plant infections and avoid the pollution caused by chemical agents.In our research,K.variicolaFH-1 could promote soybean growth and development,and induce resistance toS.sclerotioruminfection in soybean.The colonization of FH-1 on soybean roots was helpful to induce the expression of defense enzymes and related defense genes involved in plant protection.This is of great significance to soybean planting and growth.Furthermore,this study is helpful in understanding the valuable first step of the interaction betweenK.variicolaFH-1,soybean plants,andS.sclerotiorum.Further research is warranted to determine the interaction mechanism betweenK.variicolaFH-1 and soybean to cope withS.sclerotioruminfection,the metabolites of strain FH-1,related enzymes and genes,and signaling pathways.

Acknowledgements

This work was financially supported by the grants from the Inter-governmental International Cooperation Special Project of National Key R&D Program of China (2019YFE0114200),and the Natural Science Foundation Project of Science and Technology Department of Jilin Province,China (20200201215JC).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendixassociated with this paper is available on https://doi.org/10.1016/j.jia.2023.01.007