Elevation of Systemic Defense in Potato Against Alternaria Solani by a Consortium of Compatible Trichoderma Spp.

The crop loss due to phytopathogens is a serious problem affecting the entire world. To avoid economic losses due to phytopathogens synthetic chemicals have been used for years generating serious concerns about the human health and environment. Today the use of benecial microorganisms to treat phytopathogens is gaining attention. In this way, Trichoderma spp. has been used for combating plant diseases and inducing defense response in plants. With this idea in mind, in this study we evaluate the effectiveness of Trichoderma viride and T. harzianum as single as well as in combination for elevating the defense response and growth promotion activities in potato challenged with Alternaria solani. The mycelial inhibition of A. solani by T. viride and T. harzianum was recorded and compared with control. Scanning electron microscope (SEM) observation revealed the collapsed hyphae and sunken conidia of A. solani due to antagonistic activity of T. viride and T. harzianum. Induction of defense enzymes including TPC, PAL, SOD and total protein content was increased in Trichoderma spp, treated plants as compared with pathogen inoculated plants. HPLC analysis demonstrated higher production in phenolic compounds during combined application of Trichoderma spp. treated potato plants in the response of A. solani infection. Moreover, treatment with Trichoderma spp. consortium showed signicant growth promotion in potato plants comparing with the control. solani conidial germination 4 spore per against Trichoderma spp. tested against spore of solani to 1.9×10 2 spore per ml. tested against the of A. solani was to 2 spore per enzymes are PR proteins belonging to PR-2 family and important parts of plant defense against pathogen infection (Karthikeyan et al., 2006). Moreover, maize seedlings treated with T. harzianum T22 augmented the level of PR-2 proteins when plants were inoculated with Pythium ultimum (Harman et al., 2004). In the same line, β-1, 3-glucanase was induced when cucumber seedlings were treated with Trichoderma spp. inducing systemic disease resistance to pathogens (Shoresh et al., 2005). In the present work we showed a signicant increase of β-1, 3-glucanase levels when potato plants were treated with Trichoderma spp. consortium. In the results obtained in this study suggest that T. viride and T. harzianum, especially when are for elevating the defense response and growth promotion activities in potato against the phytopathogen A. solani. Therefore, these Trichoderma strains can good candidates as biological control agents to use in important agricultural crops such as potato.


Introduction
The crop loss due to numerous pests and pathogens threatens is a current serious problem worldwide. However, plants can have a complicated defense network in response to phytopathogens. The most important forms of induced resistance in plants fall into two categories such as systemic acquired resistance (SAR) and induced systemic resistance (ISR). The rst resistance, called SAR, is triggered when pathogens invade plants where the potential signal molecule salicylic acid (SA) is required and it is noted and related with PR protein accumulation for its activation (Moghaddam et al., 2019). The second defense response of induced resistance through plant growth promoting rhizobacteria, called ISR, is produced by translocatable signals and indirectly related to SA, but it is directly regulated by jasmonic acid and ethylene (Conrath, 2011). Among the most important agricultural crops for human consumption is potato (S. tuberosum L.) which has been an important mankind's food crops and constituent of cuisines. Since it is an important source of vitamin B, vitamin C and carbohydrate, it has been the third most prominent cultivated crops worldwide. Early blight caused by A. solani is a major constraint in potato production, since generates severe reduction in this plant production (Shuman and Christ, 2005) hampering crop production globally. Therefore A. solani phytopathogen has been associated with remarkable losses in terms of quantity and quality during storage (Al-Mughrabi, 2005). Among the characteristics disease symptoms rstly there are irregular dark brown to black spots in concentric rings visible on older leaves because of unequal growth of pathogen in damaged tissue (Fairchild et al., 2013). The lesions look like "target-spot" and "bull's eyes" and prominently affect the tubers and diminish the germination ability of seed potatoes.
During the years the disease has been managed by a regular application of synthetic chemicals on the seed treatment as well as foliar spray, but these are not long-term solutions since the pathogen population has developed resistance against these chemicals (Edin et  Trichoderma spp. is a soil borne genus, which has a well-established antagonistic properties, forms a green spores has wide diversity in ecosystem and can potentially colonize plant root and shoot promoting plant growth by producing bene cial metabolites (Contreras-Cornejo et al., 2014; Manganiello et al., 2018). Trichoderma spp., use their enzymatic and chemical weapons for combating the disease and induce defense response in plants. Defense signal transduction pathways have been employed to trigger the defense genes regulating the antagonistic activity. These pathways include mitogen-activated protein kinase (MAPK) cascades, heterotrimeric G-protein signaling and cAMP pathway (Zeilinger and Omann, 2007). Trichoderma spp. can induce ISR defense by the stimulation of chitinase and peroxidase activity, and induce genes that are involved in jasmonic acid or ethylene signaling pathways playing a pivotal role in to confer resistance to plant against several phytopathogens (Shoresh et  In this study we evaluate the effectiveness of T. viride and T. harzianum as single as well as in combination for elevating the defense response and growth promotion activities in potato challenged with A. solani.

Microbial strains
The culture of A. solani pathogen strain was provided from the Indian Type Culture Collection (ITCC), New Delhi, India with the ITCC No. 3640. The fungus was cultured on Potato Dextrose Agar (PDA) medium. Moreover, the antagonistic cultures of T. viride (ITCC No. 7057) and T. harzianum (ITCC No.6908) were provided from ITCC, New Delhi. The biocontrol agents were cultured on Trichoderma Selective Medium (TSM) and stored at 4°C till further use.
Antagonistic potential of Trichoderma spp. was evaluated on PDA Petri plates by dual culture assay (Fokkema, 1978). The mycelial discs of 6 mm diameter were taken from the Trichoderma spp. ve days old cultures and were placed at one end of the PDA plates keeping 1 cm distance from the edge. Mycelial discs (6 mm) of test pathogen were also placed opposite to the mycelial discs of Trichoderma spp. Control plates were maintained for both Trichoderma spp. and pathogen. These plates were incubated at 28°C and percent growth inhibition (PGI) was calculated on seventh day by using the following formula: Where, PGI = Growth inhibition of pathogen (%) C = Radial growth of the pathogen (control) T = Radial growth of the pathogen (treated).
Study of A. solani conidial germination Inhibition by Trichoderma spp. using microscopy A. solani was tested by microscopic slide technique to determine the conidial germination (Zivkovic et al, 2010). Edges of parasitized A. solani hyphae by Trichoderma spp. were taken and placed on clean slides. The cultures of Trichoderma spp. and A. solani grown on PDA medium were incubated in BOD at 28°C. Spores were harvested in Tween 20 solution (v/v 0.01%), and concentration of spore suspensions determined by hemocytometer. 50μl of Trichoderma spp. and A. solani suspensions were mixed separately, and then poured into cavity slides. In the control only conidial suspension of A. solani with sterile distilled water was taken and each slide was incubated for 24h in moist chambers. Three replications were conducted for each treatment. Inhibition of conidial germination was observed under a light microscope.
Scanning electron microscope (SEM) study of post-interaction events 1 cm bits of agar from the interaction region were taken from each plate and cultured on fresh sterile plate. The sample was vacuum dried with help of vapor diffusion dehydration assembly and xed at 4°C for 2 h in 4 % (v/v) glutaraldehyde in 0.05% sodium cacodylate buffer solution (pH 7.2), after repeated washing with same buffer the sample was dehydrated with 70-100% acetone and post xation was done with 1% (w/v) osmium tetroxide. After xation, the sample was placed in sterile separate aluminum disc cup and critical points dried in CO 2 , samples were mounted on double layer tape a xed to SEM specimen and sputter-coated with gold (Lopez-Llorca and Duncan 1988). These samples were examined at 15 kV in ZEISS electron microscope (Japan).

Preparation of Inoculum for Treatment of Potato tubers with Trichoderma spp.
Both T. viride and T. harzianum were selected and seven-day old cultures were washed properly with sterile distilled water and ltered through double layer muslin cloth and used in spore suspension preparation. The spore's quantity measured as 2×10 7 spore per ml was maintained by using a hemocytometer.
Culture of A. solani (10 days old) was used for making spore suspension of test pathogen. Culture of A. solani was ooded with Tween 20, scraped with the help of sterile rubber spatula. The test pathogen spore suspension was ltered through double layers of cheese cloth. Final spore concentration in suspension was measured by hemocytometer and adjusted to 1.5-2.0×10 5 conidia per ml to use for inoculation in potato plants.

Effect of treatments on plant growth activities in vivo
The experiments were conducted in the green house the Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, BHU, Varanasi. The details of the treatments used to determine the plant growth promotion activity are given below: The seed tubers of potato variety 'Kufri Bahar' were sown in 30 cm size pots, which were already lled with sterile soil mixture containing, sandy loam soil: farmyard manure: vermi-compost in the ratio of 2:1:1. Seed tubers were treated separately with talc-based formulation of T. viride, T. harzianum (2×10 7 spore per ml) and their mixture (1:1). The treated potato tubers were further incubated for 24 h under room temperature and ready for sowing in pot. In control pot, tubers were treated with only formulation material without Trichoderma spp. There were three replications which were conducted for one treatment with each pot containing ve seed tubers sown and maintained regularly with water. The whole experiment was repeated twice, and greenhouse conditions were maintained at 30°C and 80 % relative humidity (RH).
After 45 days of sowing, three plants were selected randomly from each treatment and growth parameters including shoot length, root length, fresh weight of shoot and root, dry weight of shoot and root, number of leaves and tuber yields were recorded. The 45 days old potato plants were used for evaluating the effect of two Trichoderma strains separately or in combination on the induction of defense activities in A. solani challenged potato plants.

Biochemical analysis of defense related enzymes
The fresh leaves were collected at 0 to 72 h after pathogen inoculation. Take plant samples from each treatment and then gently washed through running tap water and use it for estimation of changes in the action of enzymes in potato plants. The samples that were collected were stored at -80°C for further used until the completion of experiments.
Total phenolic content (TPC) assay The determination of total phenolic content was done following the method of Ragazzi and Veronese (1973). Brie y we took 0.1 g fresh leaf tissue was crushed in 10 ml of 95 % ethanol and left the sample for 1 h and centrifuged at 13,000 g for 10 min, collecting the supernatants. Enzyme solution 1 ml was taken in a separate test tube, 5 ml of distilled water and 0.5 ml of 50 % Folin-Ciocalteau's reagent (FCR) were added and gently mixed. After 10 min, 1.0 ml of sodium carbonate (5 %) was added, and the reaction mixture was vortexed and left for 1 h and to measure the absorbance at 725 nm with ethanol as black.
The absorbance values were expressed in µg gallic acid (GA) equivalent g -1 fresh weight (FW).

Phenylalanine ammonia-lyase (PAL) assay
Phenylalanine ammonia-lyase analysis was assayed following the method of Brueske (1980). Fresh leaf sample (0.1 g) was homogenized in 2 ml of 0.1 M sodium phosphate buffer (pH 7.0) which consisted on 1.4 mM β mercaptoethanol in a pre-chilled mortar and pestle, and the solution was centrifuged at 16,000 g for 15 min at 4°C. The reaction mixture, comprising 0.2 ml of enzyme extract, was incubated with 0.2 M phosphate buffer (0.5 ml; pH 8.7) followed by the addition of distilled water 1.3 ml . The reaction started by the adding of 0.1 M phenylalanine and incubated for 30 min at 32°C. The reaction was stopped by addition of 1M trichloroacetic acid 0.5 ml and absorbance was recorded at 290 nm. The amount of trans-cinnamic acid was de ned as the µmol TCA mg -1 fresh weight.

Total Protein Content
The method developed by Lowry et al. (1951) was used to determine the total protein content in potato leaves. Fresh leaf sample 1 g from each treatment was homogenized in extraction buffer in pre-chilled mortar and pestle. Enzyme extract was centrifuged at 10,000 g for 30 min at 4°C collecting the supernatants. 7.5 ml of supernatant were transferred in a separate tube, gently mixed with sample buffer 2.5 ml and used for protein estimation. The working standard solution was pipette out and 0.2, 0.4, 0.6, 0.8 and 1.0 ml were put into series of test tubes affording to 1 ml nal volume with distilled water. A separate tube with 1 ml of water was performed as a blank. 5 ml of solution C were added in each tube and incubated at room temperature for 15 min and then 0.5 ml FCR were added and gently mixed and incubated 30 min in dark condition. Absorbance was recorded at 660 nm against the blank and amount of total protein was expressed as µg g -1 fresh weight.

Superoxide Dismutase (SOD) assay
Superoxide Dismutase (SOD) activity was performed following the Fridovich method (1974). 0.5 g fresh leaf sample were taken from each treatment and homogenized in 2 ml of 0.1M phosphate buffer containing 0.5 mM EDTA at pH 7.5. The homogenate was centrifuged at 15,000 g in 4°C for 20 min. The reaction mixture comprised methionine 200 mM, phosphate buffer 100 mM (7.8 pH), nitroblue tetrazolium chloride 2.25 mM, EDTA 3.0 mM, sodium carbonate 1.5 M and enzyme extract, making up 3 ml of reaction mixture nal volume. The reaction was begun by adding 2 mM ribo avin (0.4 ml), and placing each tube under 15 W uorescent lamps for 15 to 20 min. The reaction was stopped by switching off the lights and keeping the tubes in dark condition for 15 min and absorbance was observed at 560 nm. Reaction mixture without enzyme was performed as a control. SOD activity was de ned as the amount of the enzyme reducing the absorbance to 50 % in comparison with control.

β-1, 3 glucanase assay
The β-1, 3 glucanase enzyme was determined following the method of Pan and co-workers (1991). Fresh leaf samples 0.1 g was homogenized in 0.05 M sodium acetate buffer 2.0 ml (pH 5.0) and the sample was centrifuged at 13,000 g at 4°C for 15 min. The reaction was started by mixing supernatant 0.25 ml, 1 M sodium acetate buffer 0.3 ml (pH 5.3) and laminarin 0.5 ml and after solution was incubated at 40°C for 60 min. The reaction is stopped by adding 3, 5dinitrosalicylic acid 0.375 ml. The nal-colored solution was diluted with distilled water and absorbance was measured at 500 nm. The β-1, 3 glucanase enzyme activity was de ned as µg glucose released min -1 g -1 fresh weight. High performance liquid chromatography (HPLC) analysis of phenylpropanoid derivatives Take 1 g of fresh leaf sample harvested 0 h, 24 h, 48 h, and 72 h after inoculation of the pathogen and was homogenized with 10 ml of 50% methanol and centrifuged for 15 min at 13,000 g. After centrifugation solvent was removed under reduced pressure on rotary evaporator (Eyela N-N series, Japan). The residue was dissolved with HPLC grade methanol. The compounds separation was done using the protocol of Singh et al., (2009). The solvent ow rate was 1.0 mL min -1 and phenolics were detected using UV detector SPD-10A.Their identi cation was done by comparing the retention times with those from authentic standards at 254 nm.

Measurement of Disease Severity
The disease severity observation was recorded after 15 days of pathogen inoculation and compared with control plants. Three leaves were randomly selected from each treatment for disease severity measurement using 0-9 scale as described by Mayee and Datar, 1986.

Statistical analysis
The analysis was done by using IBM SPSS, Version 20. The presented values from different experiments were the mean of three replications and ± show standard deviation (SD) for each treatment. The present data was statistically analyzed by using variance one-way analysis (ANOVA) and treatment mean values were compared with Duncan's multiple range tests at the p ≤ 0.05 signi cance level.

Results
Growth inhibition of pathogen by Trichoderma spp. in dual culture assay The dual culture assay results revealed that both T. viride and T. harzianum signi cantly inhibited the growth of A. solani under in vitro condition ( Figure 1). As shown in gure 1, maximum growth inhibition was recorded by T. viride against A. solani (91.88%) on seven day of inoculation, while for T. harzianum, the lowest growth inhibition against A. solani (80.11%) as compared to control.

Study of A. solani conidial germination Inhibition by Trichoderma spp. using microscopy
A study of A. solani conidial germination inhibition assay by T. viride and T. harzianum was realized using microscopic slide technique. In general, both Trichoderma spp. signi cantly reduced A. solani conidial germination when compared to the control. The initial spore's concentration in the control suspension of A. solani was found to be 4.5×10 4 spore per ml against Trichoderma spp. The highest reduction of A. solani conidial germination was found when they were tested against T. viride, being the spore concentration of A. solani reduced up to 1.9×10 2 spore per ml. Whereas, when the pathogen was tested against T.
harzianum the spore concentration of A. solani was slightly reduced to 3.31×10 2 spore per ml.
Ultrastructure defects on A. solani hyphae and spores under Scanning electron microscope Scanning electron microscopy was used to identify the morphological changes in A. solani hyphae and spores due to antagonistic activity of T. viride and T. harzianum ( Figure 2 Figure 5). Microscopic visualization of superoxide radicals in potato leaves revealed that minimum reactive oxygen species (ROS) accumulation was found in control as compared to other treatments. However, higher production of ROS was found in the plant treated with the consortium of Trichoderma spp. Moreover, when plants were treated with a single Trichoderma spp. they have less production of ROS as compared to combinations of both Trichoderma spp. (Figure 6).  (Table 2).  T1  T2  T3  T4  T1  T2  T3  T4  T1  T2  T3  T4  T1  T2  T3  The total protein content was measured in all treatments and the results showed the plants treated with Trichoderma spp. Consortium triggered the total protein content in the leaves of potato increasing trend 0 to 48 hapi ( Figure 10). Maximum protein content was noted to be nearly 2.50 times higher in treatments with Trichoderma spp. consortium and 1.82 times and 1.86 times higher in treatments with T. viride and T. harzianum respectively at 48 h after inoculation of the pathogen compared to control. At 72 hapi total protein content declined sharply in all treatments ( Figure 10). Similarly, the superoxide dismutase (SOD) activity was measured and the results showed an elevated level of SOD activity in plants treated with Trichoderma spp. consortia, challenged with A. solani exhibiting a signi cantly higher content of SOD than pathogen inoculated control plants ( Figure 11). Individual application of T. viride and T. harzianum remarkably increased level of SOD observed. After 48 hapi the level of SOD was declining gradually.
Effect of Trichoderma spp. on activity of β-1, 3-glucanase Pathogenesis related protein (PR-2) also is correlated with β-1,3glucanase enzyme activity providing rst line defense against pathogen infection. Therefore, the β-1, 3 glucanase enzyme activity was measured and the results showed that this activity is signi cantly higher in the consortium of T. viride + T. harzianum treated plants as well as individually treated with Trichoderma spp. compared to the control plant ( Figure 12) being the maximum β-1, 3-glucanase level at 72 h after pathogen inoculation, that is, 35.51 µg glucose released min -1 g -1 fresh weight, it means 2.21-fold higher than control. The plants treated with singly T.
viride and T. harzianum was recorded 25.15 µg glucose released min -1 g -1 fresh weight and 23.71 µg glucose released min -1 g -1 fresh weight respectively that were, 1.56 and 1.47-fold higher than control which contains the lowest value of β-1,3-glucanase, that is, 16.04 µg glucose released min -1 g -1 fresh weight ( Figure 12). Plants that are treated with bioagents can augment their effector molecules activity of the ux by phenylpropanoid pathway. PAL is the rst line defense enzyme that is required for the crucial phenylpropanoid biosynthesis pathway, leading to synthesis of potential phytoalexins, which sharp capability in plants for ghting against infamous pathogens (Nicholson and Hammerschmidt, 1992). There is an increase level of mRNA that encodes the chalcone synthase and PAL in the early stage of plant-antagonistic microbes' interaction (Zodor and Anderson, 1992 Phenolic compounds are found naturally in living plants that provide support from invasion of pathogenic fungi and play crucial a role in biosynthesis of cinnamic acid, phenylalanine and phenylpropanoids. Singh and co-workers (2011) identi ed some potential phenolic compounds such as, ferulic acid, tchlorogenic acid and protocatechuic acid which provide forti ed defense against invaders,. The results of the present work showed that phenolic compounds, such as gallic acid, shikimic acid and syringic acid, were increase by the treatment of Trichoderma spp. consortium. Gallic acid is the important source of antifungal and converted into gallotannins, which showed antimicrobial properties (Salisbury and Ross, 1986). Moreover, the shikimic acid pathway may regulate the biosynthesis and accumulation of potential plant phenolic compounds, which are good precursors of lignin and tannins avonoids (Shoresh et al., 2005).

Discussion
The crucial superoxide dismutase (SOD) enzyme directly is involved in the activation of ascorbate-glutathione pathway to enhance the scavenging of free radicals (Hernandez et al., 2011). Under biotic and abiotic stress conditions, plants produce huge amounts of ROS that can lead the scavenging capacity and damaged the plant cellular system through lipid peroxidation (Mittler, 2002). SOD worked as a denature enzyme and catalyzed the huge production of O 2 and H 2 O 2 from superoxide radicals. Proteomic analysis revealed that the Trichoderma spp. treated roots can enhance the augmentation of SOD and other detoxifying enzymes that protect from collar rot pathogen infection (Smirnoff, 1993). In this work we show that potato plants treated with Trichoderma spp. consortium have a high production of SOD enhancing ROS scavengers and therefore, protecting the potato plants against A. solani.
Plant β-1, 3-glucanase enzymes are PR proteins belonging to PR-2 family and important parts of plant defense against pathogen infection (Karthikeyan et al., 2006). Moreover, maize seedlings treated with T. harzianum T22 augmented the level of PR-2 proteins when plants were inoculated with Pythium ultimum (Harman et al., 2004). In the same line, β-1, 3-glucanase was induced when cucumber seedlings were treated with Trichoderma spp. inducing systemic disease resistance to pathogens (Shoresh et al., 2005). In the present work we showed a signi cant increase of β-1, 3-glucanase levels when potato plants were treated with Trichoderma spp. consortium.
In summary the results obtained in this study suggest that T. viride and T. harzianum, especially when both are applied together, are effective for elevating the defense response and growth promotion activities in potato against the phytopathogen A. solani. Therefore, these Trichoderma strains can be good candidates as biological control agents to use in important agricultural crops such as potato.

Declarations
Funding SK, RC, LB and CK are grateful to Banaras Hindu University, Varanasi, India for nancial support.

Con icts of interest/Competing interests
The authors declare that there is no con ict of interest/ competing interests whatsoever.
Availability of data and material (data transparency) The data would be made available on demand basis.
Code availability (software application or custom code)

Not applicable
Authors' contributions (optional: please review the submission guidelines from the journal whether statements are mandatory) SK, CK, RC and ES were involved in the idea generation. SK, LB, CK conducted the experiments and analyzed the data. SK, CK, ES were involved in manuscript preparation and editing. RC supervised the work.
Additional declarations for articles in life science journals that report the results of studies involving humans and/or animals         Effect of Trichoderma spp. on PAL activity in potato leaves challenged with pathogen at different time intervals.

Figure 10
Total protein content at different time of intervals potato plants treated with Trichoderma spp. either individual of consortium and challenged with A. solani.

Figure 11
Effect of Trichoderma spp. and their consortium on SOD activity (unit g-1 FW) in potato leaves against A. solani at different time of intervals challenged with pathogen.

Figure 12
Effect of individual or consortium of Trichoderma spp. on β-1,3-glucanase activity (µg glucose released min-1 g-1 fresh weight) in potato leaves at different time of intervals challenged with pathogen.