Physiological Analysis Reveals the Possible Resistance Mechanisms of Glycine max to Fusarium solani

Sudden death syndrome (SDS) of soybean is a complex root rot disease caused by the semi-biotrophic fungus Fusarium solani (F. solani) and a leaf scorch disease; caused by toxins produced by pathogen in the roots. However, the mechanism of soybean resistant to F. solani is still poorly understood. Eighteen soybean cultivars were screened for SDS resistance, with one cultivar showing susceptibility and one cultivar showing resistance to F. solani infection. Histochemical analysis with diaminobenzidine (DAB) and Trypan blue staining indicated an accumulation of reactive oxygen species (ROS) and cell death in surrounding area of SDS which was higher in susceptible cultivar than in resistant cultivar. Furthermore, exogenous salicylic acid (SA) application also induced some level of resistance to F. solani by the susceptible cultivar. A biochemical study revealed that the activities of superoxide dismutase (SOD), peroxidase (POD), and enzymes involved in scavenging ROS, increased in susceptible cultivar after SDS infection. In addition, hydrogen peroxide (H2O2) and malondialdehyde (MDA) content also increased in the susceptible cultivar than in resistant cultivar. High-performance liquid chromatography (HPLC) analysis indicated that free and total salicylic acid (SA) content increased in the susceptible cultivar than in resistant cultivar. In addition, a real-time quantitative PCR analysis showed an accumulation of pathogen related (PR) genes in the resistant cultivar than in susceptible cultivar. Our results show that (i). F. solani infection can increase endogenous SA levels, antioxidase activities, ROS and cell death in susceptible soybean cultivar to induce resistance against Fusarium solani. (ii). F. solani infection induced the expression of SA marker genes in resistant soybean cultivar to enhance resistance.


Introduction
Soybean production is often affected by many diseases in major cropping areas (Wrather et al., 2010).Sudden death syndrome (SDS) caused by Fusaruim solani (F.solani) occurs frequently in the top eight soybean producing countries in the world (Wrather et al., 2010).SDS is associated with root rot, vascular discoloration of stems, root chlorosis and necrosis, defoliation and plant death (Roy et al., 1997).Mostly, SDS is more easily detected in soybean fields after flowering, when the leaves show interveinal choroids and necrosis.Meanwhile, SDS can be expressed in severe and mild forms.The occurrence and geographical distribution of pathogens causing the latter is unknown (Scandiani et al., 2011).
Exposure of plants to unfavorable conditions, makes them develop an integrated defense mechanism against fungal diseases which include chemical and physical barriers; and inducible defense (Dixon et al., 1994).So far as resistance responses other than susceptibility and immunity is concerned, the invasion of plant by fungal hyphae is likely to induce and sustain expression of some plant defense-related genes.Induced defenses attempts to prevent or reduce pathogen access by activating molecules that are antimicrobial, antioxidants, involved in the SA signaling pathways (Lamb & Dixon, 1997).Phytohormones play important roles in regulating developmental process and signaling networks which are involved in plant response to a wide range of abiotic and biotic stresses (Robert-Seilaniantz et al., 2007;Bari & Jones, 2009).Three major signaling molecules, Salicylic acid (SA), Jasmonic acid (JA) and Ethylene (ET) are recognized as major defense hormones against various pathogens (Glazebrook et al., 2003;De et al., 2005;Koornneef & Pieterse, 2008).SA is associated with resistance against biotrophic and hemibiotrophic pathogens, and with triggering systematic acquired resistance (SAR) in many species including Arabidopsis thaliana and wheat (Triticum aestivum) (Görlach et al. 1996).Induction of SAR is accompanied by accumulation of SA and up-regulation of a set of genes encoding pathogenesis-related (PR) proteins in dicot plants such as tobacco (Nicotiana tabacum) and Arabidopsis thaliana providing a wide range of protection against pathogens (Ward et al., 1991;Uknes et al., 1992).The elevated expression of defense genes have been assumed as a molecular evidence of induced resistance (Sumayo et al., 2014).Natriuretic Peptide Receptor 1 (NPR1) gene is a key regulator of the SA signaling pathways (Yan & Dong, 2014).Enhanced Disease Susceptibility 1 (EDS1) gene was important for mediating resistance to a broad range of pathogens (viral, bacterial and fungal pathogens) yet showed specificity to the class of resistant R genes that it affected (Hu et al., 2005).The EDS1 protein has been found to be complex with both the pathogen effectors and their cognate proteins and partitioning of the EDS1 complex in the cytoplasm nucleus is required for full activation of local resistance (Zheng & Dong, 2013).EDS1 is required to induce SA biosynthesis (Zheng & Dong, 2013).Also, Chitosan which is used in agriculture in seed treatment and biopesticide helping plants to fight off against fungal infections, induced a significant increase in the activities of polyphenoloxidase, peroxidase, and enhanced the content of phenolic compounds in tomato fruits, thus providing protection against gray mould and blue mould diseases (Liu et al., 2007).Plants resistance can be induced by application of synthetic compounds such as functional analogs of SA, for example benzothiadiazole-7-carbothioic acid (acibenzolar-S-methyl) or benzothiadiazole (BTH).It has been shown that BTH which is a nontoxic compound, induced systematic resistance by exogenous root-treatment in tomato and controlled crown and root rot caused by F.oxysporum radices-lycopersici (Benhamou & Bélanger, 1998).Fusarium wilt of tomato was effectively controlled by foliar spray of validamycin A or validoxylamine A, which induced SA accumulation and development of systematic resistance (Ishikawa et al., 2005).Exogenous application of SA induces plant resistance to different kinds of pathogens that are associated with oxidative burst, cell wall enforcement and up-or down-regulation of gene expression (Oostendorp et al., 2001).
Production of ROS such as the superoxide anion (O 2 .-) and hydrogen peroxide (H 2 O 2 ), as one of the earliest response to pathogen attack, can trigger hypersensitive cell death.Abnormally high production of ROS causes damage to biomolecules, whereas ROS at moderate concentrations act as a second messenger in signal cascades that mediate several responses in plant cells including program cell death (Sharma et al., 2012).The hypersensitive response (HR), as an early defense response, restricts pathogen infection to the site of attempted ingress by necrosis and cell death.Plants have an efficient antioxidative, enzymatic and non-enzymatic protective mechanisms to scavenge excess ROS.Several antioxidative enzymes including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) are involved in detoxification of ROS (Zhang et al., 1995;D. H. Lee & C. B. Lee, 2000).
Although much efforts have been made to identify mechanisms of resistance against SDS, much remains to be elucidated about the physiological and molecular capabilities of the soybean plant against F. solani infection.
Here we performed a comparative study between resistant and susceptible soybean cultivars to distinguish the effects of some antioxidant and biosynthetic enzymes in response to F. solani infection.We also investigated the effect of exogenous application of SA, as a key factor in SAR, on soybean resistance to F. solani infection.Finally, to identify possible PR genes involved in the resistance process between the resistant and susceptible cultivar, RT-qPCR analysis was performed.

Plant Materials and Chemicals
Eighteen soybean cultivars used in the study were provided by Key Laboratory for Crop Genetics and Breeding of Sichuan Agricultural University, China.Seeds were surface-sterilized for 20 min in a 20% solution of sodium hypochlorite and then rinsed three times with sterilized water.Seeds were grown in an 11.5 cm diameter paper cups filled with perlite and placed in a climatically grown chamber.Conditions in the growth chamber included day/night at a regulated temperature of 25 ºС.Watering was done as required to promote seed germination.

Fungal Growth and Inoculation
The fungi Fusarium solani isolate 2hao3 was provided by the Key Laboratory for Major Crop Diseases of Sichuan Agricultural University, China.Isolate was obtained from soybean roots and preserved on potato dextrose agar (PDA) prior to sub cultures.2 mm plug from the stock culture was used for sub cultures for inoculation by placing fungal isolate on PDA medium in petri dishes.The fungus was incubated at a temperature of 28 ºС for 7 days in the dark in a growth chamber.The seedlings were removed from the perlite after 3-day growth period and washed with distilled water prior to inoculation.Inoculation was performed by using the hypocotyl inoculation method described previously by (Haas & Buzzell, 1976).Disease development was observed for 2 days.Control plants were inoculated with 7 days grown PDA on a petri dish without fungus.Fungal inoculated and non-fungal inoculated seedlings were placed in a growth chamber at a temperature range of about 25 ºС without light condition.

Screening and Disease Assessment
Assessment of disease severity on all the 18 soybean cultivars was done according to (Ishikawa et al., 2005) with some modifications.At 2 days post inoculation (dpi) by F. solani the disease index (on 1-5 scale) on each plant was recorded according to vascular browning and the mean value of 10 plants from each cultivar calculated for disease severity.For evaluation of vascular browning, the basal stems were cut and vascular browning was rated on a scale of 1-5; where 1 = no symptoms or vascular browning; 2 = 1-25% vascular; 3 = 6-50% vascular browning; 4 = 51-75% vascular browning; 5 = more than 75% vascular browning.The mean value recorded for Nandou12 was 1 = no symptoms and Juiyuehuang recorded 5 = more than 75% vascular browning (Figure 1).These formed our basis for selecting Nandou12 as resistant and Juiyuehuang as susceptible cultivars.

Exogenous SA Application
To determine whether exogenous SA application can induce systematic acquired resistance in soybean, we first sprayed (For 48 h: at every 6 h intervals) the seedlings with SA which was dissolved in deionized water at a concentration of 100 µM and 200 µM in order to compare the disease development to seedlings inoculated with fungus without exogenous SA application.This assay was conducted as previously described (Spletzer & Enyedi, 1999).

Reverse Transcription and Quantitative PCR (RT-qPCR) Analysis
Total RNA was extracted using a plant total RNA Miniprep purification Kit (Tiangen, http://www.tiangen.com/)cDNA was reversely transcribed from 2 µg of total RNA using an oligo dT 20 primer and MLV reverse transcriptase (http://www.invitrogen.com).First strand cDNAs of reversely transcribed 50ng of RNA was used for semi-quatitative RT-PCR analysis Extaq DNA polymerase (TaKaRa) and qPCR with Universal SYBR ® GREEN qPCR Master Mix (2×) (Gangchi Bio).The parameters of the semi-quantitative PCR were as follows: 95 ºC for 5 min, 95 ºC for 15 s, 50 ºC for 30 s, 72 ºC for 1kb min -1 , and another cycle (step 2) was repeated according to the gene expression level of the specific genes.Parameters of the qPCR were as follows 95 ºC for 3 min, 95 ºC for 15 s and 55 ºC for 15 s, and 72 ºC for 20 s, go to step 2 for 39 more cycles.Then increment of 0.5 ºC from 65 ºC to 95 ºC for 5 seconds was used for melt curve analysis.ΔΔCq method was used to normalize the qPCR data according to (Du et al., 2016).GmACT3 (Glyma09g17040) was amplified as an internal control.Gene-specific primer pairs were designed using Primer 5.0 (Table 1).

Endogenous SA Measurement
SA was extracted and measured according to modification from previous studies (Wang et al., 2011).200 mg of soybean tissue was ground to fine powder with N 2 and extracted once with 1.5 ml of 90% methanol followed by one extraction with 1.5 ml of 100% methanol.The methanol fraction was equally split into two micro centrifuge tubes (for total and free SA analyses, respectively) and dried in the fume hood overnight.The pellet was dissolved by adding 500 μl of 100 mM sodium acetate (pH 5.5).To half of the duplicated samples, 40 μl of β-glucosidase (Sangon, A662003-0010) were added to digest glucosyl-conjugated SA (total SA) for 1.5 h at 37 °C.(About 80 units/g fresh weight).All the samples were treated with an equal volume of 10% trichloroacetic acid (TCA) and centrifuged at 10,000g for 10 min.The supernatant was extracted twice with 1 ml of extraction solvent (ethylacetate: cyclopentane: 2-propanol 100:99:1, v/v).The top (organic) phase was collected in a micro centrifuge tube and dried in a fume hood overnight.The residual fraction was re-suspended in 0.5 ml of 55% methanol by vortex and was passed through a 0.2-um nylon spin-prep membrane (Fisher 07-200-389) via centrifugation for 2 min (14,000 g) before being subjected to HPLC analysis.A Dionex AS50 HPLC instrument with an Acclaim 120C18 reverse column (4.6 × 250 mm) and an RF2000 fluorescence detector was used to separate and detect SA.The mobile phase included a gradient of methanol and 0.5% acetic acid.SA was detected at 6.5 min with 301-nm excitation/412-nm emission.The standard curve was made from quantification of SA at concentration of 10, 8, 6, 2, and 1 mg mL -1 and used to calculate the final concentration in each sample using Microsoft Excel software.

Determination of Antioxidant Enzymatic Activity
The enzymes activity was carried out by grinding 0.5 g of 2 days infected soybean tissues with 2 mL ice-cold 25 mM HEPES buffer (pH 7.8) containing 0.2 mM, EDTA, 2 mM ascorbate and 2% PVP.Further, the homogenates were centrifuged at 4 ºC for 15 minutes at 13,000 g and the supernatant was used for enzyme activities analysis.All the various steps in the preparation of the enzyme extract were carried out at 4 °C.Peroxidase (POD) activity was measured according (Egley et al., 1983), the total volume of 2 mL mixture contained 25 mM (Ph 7.0) sodium phosphate buffer, 0.1 mM EDTA, 5% guaiacol (2-ethoxyphenol), 1.0 mM H 2 O 2 and 100 μl enzyme extract.Superoxide dismutase (SOD) activity was also measured as previously described (Giannopolitis and Ries 2010).

H 2 O 2 Measurement
Hydrogen peroxide H 2 O 2 content from the 2days infected soybean tissues was measured according to (Du et al., 2011).About 1 g of soybean hypocotyl was homogenized in an ice bath with 5 ml 0.1% (w/v) TCA.The homogenate was transferred into a tube and centrifuged at 12,000 g for 20 min at 4 ºС.0.5ml of the supernatant was added to 0.5 ml 10 mm potassium phosphate buffer (Ph.7.0) and 1 ml potassium iodide (KI).The absorbance of supernatant was read at 390 nm.H 2 O 2 content was determined by a standard curve.

Malondialdehyde (MDA) Content Measurement
MDA content of the 2days infected soybean tissues was quantified according to (Sun et al., 2006;Qian et al., 2007).About 1 g of soybean tissues (hypocotyl) was homogenized with 5% trichloroacetic acid (TCA) on ice and centrifuged at 3,000 g for 10 min at 4 ºС. 2 mL of the supernatants was transferred into another tube added with 2 ml 0.67% thiobarbituric and incubated at 100 ºС for 15 min.The cooled mixture was centrifuged at 4,000 g for 10min.The supernatants were subjected to analysis at 450 nm (A 450 ) 532 nm 600 nm (A 600 ) in spectrometer.The amount of MDA was calculated using an extinction coefficient of 155 Nm -1 cm -1 and according to the formula: MDA (µmol L -1 ) = 6.45× (A 532-A 600 ) -0.56×A 450.

Statistical Analysis
All experiments were repeated three times, with three replication each.Statistical calculations were performed using SPSS-20 (SPSS, Chicago, IL, USA).For disease severity assessment, a minimum of ten plants were evaluated for each replicate.Tests for significant difference among physiological parameters under different treatment were conducted using analysis of variance (ANOVA) with mean separation using Duncan's multiple range tests (DMRT) at the 0.05 level of confidence.

Phenotypic Expression of Soybean Seedlings to F. solani Infection
In the current study, 18 soybean cultivars were evaluated for SDS resistance to F. solani infection.Inoculated seedlings were monitored and disease symptoms were recorded at 2dpi.Jiuyuehuang showed severe disease symptoms under F. solani infection whiles Nandou12 showed no disease symptoms (Figure 1; Figure 2: JT and NT).Once the symptoms of the disease appeared, fungal inoculated and non-fungal inoculated plants were collected at 2 dpi.The appearance of the symptoms provided a confirmation that the pathogen had penetrated the host tissues and infection was successful.Visual disease assessment (VDS) was used to evaluate the resistance level between the two soybean cultivars.See Materials and Methods section for details.

Histo-c
Results fro cell death Through s Likewise c Figure 3. Jiuyuehu

Detect
The effect was invest F. solani i (Figure 5).(Luo et al., 2011).SA pathways is often induced by pathogen infection and is effective in mediating resistance against biotrophic pathogens (Thaler et al., 2012).SA signaling was shown to be important in defense against F. graminaruim infection in A. thaliana and in wheat (Makandar et al., 2006;Makandar et al., 2010;Makandar et al., 2012).From the current study, endogenous SA content (Total and conjugated SA) increased in Jiuyuehuang than in Nadou12 which suggests the accumulation of SA as a result of the fungal infection.
The result from the gene expression analysis indicated that more defense genes were expressed in Nandou12 than in Jiuyuehuang.The expression of NPR1 was also up-regulated in Nandou12 than in Jiuyuehuang indicating that the gene expression involved in SA signaling pathways was up-regulated in Nandou12 than in Jiuyuehuang.We suggest from the current result that in the event of fungal stress in soybean, SA mediated disease resistance plays an important role in Nandou12 resistance to F. solani.
In addition, results from the current study also showed that there was an increase in Peroxidase (POD), superoxide dismutase (SOD) and malondialdehyde (MDA) as a result of the fungal stress (Figure 7).SOD is one important enzyme in ROS metabolism and catalyzes the dismutation of oxygen (O 2 )-and hydrogen peroxide (H 2 O 2 ) (Gill & Tuteja, 2010).Increased and decrease in SOD activity have frequently been correlated with disease resistance and susceptibility (Yang et al., 2003).Vanacker et al. (1998) reported that an increase in SOD activity following pathogen attack might be required to catalyze the synthesis of H 2 O 2 during the oxidative burst and to prevent the accumulation of superoxide.The current study recorded an increase in SOD activity in the fungal infected plants of Nandou12 more than the fungal infected plants of Jiuyuehuang.Similar result was recorded in the POD level with increased activity in the fungal infected plants of the resistant cultivar than in fungal infected plants of the susceptible cultivar.This suggests that antioxidant defense mechanism activated under stress remained operative throughout that challenging period, enabling plants to adopt to such conditions (Pérez-Clemente et al., 2012).MDA is one final decomposition product of lipid peroxidation and has been used as an index for the presence of lipid peroxidation (Esim et al., 2012).While more ROS was accumulated, more MDA was also accumulated in Nandou12 (Figures 3d and 7c) respectively.Which we conclude that the fungal stress had more damaging effect on Jiuyuehuang more than the Nandou12.MDA content increased in water stressed olive plants which has resemblance with our results (Sofo et al., 2004).The increased level of activated oxygen species could contribute to the symptoms development and pathogenesis in compatible plant-virus interactions.In the current study, the higher level in ROS is consistent with the increase in H 2 O 2 activity (Xi et al., 2007).

Conclusion
In conclusion, F. solani infection revealed disease symptoms in Jiuyuehuang with no disease symptoms in Nandou12.The plant cells stained by DAB and Trypan blue produced a high amount of ROS and cell death in the area of infection respectively.Enzymatic and non-enzymatic antioxidative pathways which are involved in the production of signaling molecules were increased in Jiuyuehuang.More PR genes were induced in Nandou12.The current findings suggest that these differences are associated with resistance.SA treatment of both cultivars rendered them more resistant to F. solani.The current results can provide novel insights for better recognition of the responsible mechanisms needed to regulate SDS resistance in soybean.The direct effect of SA should further be examined on the above-mentioned enzyme activities and their gene expression in conferring resistance to pathogens.Exogenous SA application was able to induce SAR against F. solani of which we propose that SA played this role through regulation of the plant antioxidative system or through the genes involve in the SA signaling pathways.