Pretreatment with Trichoderma harzianum alleviates waterlogging-induced growth alterations in tomato seedlings by modulating physiological, biochemical, and molecular mechanisms

Journal Pre-proof Pretreatment with Trichoderma harzianum alleviates waterlogging-induced growth alterations in tomato seedlings by modulating physiological, biochemical, and molecular mechanisms Amr A. Elkelish, Haifa Abdulaziz S. Alhaithloul, Sameer H. Qari, Mona H. Soliman, Mirza Hasanuzzaman

PII:

S0098-8472(19)31543-6

DOI:

https://doi.org/10.1016/j.envexpbot.2019.103946

Reference:

EEB 103946

To appear in:

Environmental and Experimental Botany

Received Date:

14 September 2019

Revised Date:

20 October 2019

Accepted Date:

9 November 2019

Please cite this article as: Elkelish AA, Alhaithloul HAS, Qari SH, Soliman MH, Hasanuzzaman M, Pretreatment with Trichoderma harzianum alleviates waterlogging-induced growth alterations in tomato seedlings by modulating physiological, biochemical, and molecular mechanisms, Environmental and Experimental Botany (2019), doi: https://doi.org/10.1016/j.envexpbot.2019.103946

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Pretreatment with Trichoderma harzianum alleviates waterlogging-induced growth alterations in tomato seedlings by modulating physiological, biochemical, and molecular mechanisms

Amr A. Elkelish1, Haifa Abdulaziz S. Alhaithloul2, Sameer H. Qari3, Mona H. Soliman4,5* and Mirza Hasanuzzaman6*,

Botany Department, Faculty of Science, Suez Canal University, Ismailia, Egypt

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Biology Department, College of Science, Jouf University, Sakaka 2014, Saudi Arabia

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Biology Department, Aljumum, University College, Umm Al-Qura University, Saudi Arabia

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Biology Department, Faculty of Science, Taibah University, Al-Sharm, Yanbu El-Bahr,

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Yanbu 46429, Kingdom of Saudi Arabia

Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza 12613,

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Egypt

Department of Agronomy, Faculty of Agriculture, Sher-e-Bangla Agricultural University,

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Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh

*Corresponding authors, E-mails: [email protected] [M.H.S.]; [email protected]

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[M.H.]

Highlights 

Waterlogging (WL) inhibited plant growth and oxidative stress

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Trichoderma harzianum (TH) improved plant physiology and metabolism under WL

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TH maintains plant nutrient status and improves the content of photosynthetic pigments under WL 1

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TH upregulates antioxidant defense system and decreases oxidative stress under WL

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TH upregulates gene expression related to sugar and alcohol metabolism as well as aquaporin

Abstract

We studied the role of Trichoderma harzianum (TH) in improving the physiological,

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biochemical, hormonal, and molecular parameters of tomato seedlings grown under waterlogging (WL, for 14 and 28 days).pretreated with. Pretreatment with TH significantly improved the growth of tomato by enhancing the chlorophyll synthesis and uptake of

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essential ions, including nitrogen, phosphorus, and potassium. A reduction in anthocyanin

content was also ameliorated significantly by TH pretreatment. TH significantly mitigated the

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WL-induced decline in height and in fresh and dry biomass accumulation. Accumulation of

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proline, flavonoids, anthocyanin, sugars, and soluble protein, increased with TH pretreatment. At both growth periods (14 and 28 days after treatment [DAT]), the accumulation of secondary metabolites, including total phenols and flavonoids, and the redox components

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(tocopherols) was increased significantly by TH pretreatment. Increased synthesis of metabolites maintained the antioxidant status of tomato, resulting in amelioration of WLinduced oxidative effects on membranes. WL and TH treatments significantly increased

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ethylene production and decreased abscisic acid content at both growth periods. The accumulation of reactive oxygen species, like hydrogen peroxide, in TH treated seedlings was correlated with the upregulation of the Fe-SOD gene. WL stress triggered the activity of sucrose synthase (SUS), lactate dehydrogenase (LDH), and pyruvate decarboxylase (PDC), which reached a maximum at 14 DAT, and TH pretreatment resulted in further enhancement above control and WL-stressed levels. Quantitative RT-PCR revealed differential expression 2

of genes, where Fe-SOD and ADH were upregulated due to TH treatment and ARE, ACO, ERF, and aquaporin were downregulated relative to control plants. Pretreatment of tomato seedlings with TH improved tolerance to WL by maintaining the antioxidant status, sugar metabolism, and expression of critical genes. These results suggest that TH pretreatment is an effective way to improve WL tolerance in tomato at vegetative stage.

Keywords: Anoxia; Hypoxia; Oxidative Stress; Osmolyte; Sugar metabolism; Gene

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expression; Trichoderma harzianum

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1. Introduction

Climate change is causing the heavy rainfall and overflow of rivers which has been

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considered a primary reason for waterlogging (WL) in crop plants (Milly et al., 2002; Wright

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et al., 2017). The resulting hypoxic or anoxic conditions in soil due to WL is one of the main factors affecting plant developmental stages and survival, and therefore crop production (Jackson and Colmer 2005; Paul et al. 2016). Plants under flooding stress develop a set of

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morphological, physiological, anatomical, and biochemical adaptations to resist the damaging effects of the low oxygen content in flooded soils (Chen et al., 2016; Ravanbakhsh et al., 2017; Anee et al. 2019).

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Soil WL decreases (hypoxic) or completely depletes (anoxic) soil oxygen levels, creating conditions that are very detrimental to normal plant development (Armstrong et al., 2000; Liu et al. 2020). Oxygen deficiency negatively influences aerobic respiration, causing reductions in plant growth by disrupting photosynthesis, hormonal balance, and nutrient uptake; in turn, growth is stunted and yields are reduced (Valliyodan et al., 2016; Liu et al. 2020). WL also induces stomatal closure, which limits CO2 availability for photosynthetic 3

carbon metabolism and subsequently induces oxidative stress due to photosynthetic free radical accumulation (Sairam et al., 2009). Programmed cell death is also related to WL and leads to formation of aerenchyma and ethylene generation (Lenochová et al., 2009; Yin et al., 2010). Waterlogging also elicits changes in developmental programs via regulation of gene expression and adaptive mechanisms, such as the development of adventitious roots (Chen et al., 2016; Song et al., 2017) and the regulation of ion uptake (Carter et al., 2006; Colmer and Flowers, 2008), aimed at mitigating flooding stress (Chen et al., 2017; Li et al., 2018).

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Modifications of the root system play a key role in withstanding WL effects; for example, accumulation of lignin and suberin at the cell level, as well as the formation of the

adventitious root system in tomato and aerenchyma development in wheat, have been

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reported in response to WL (Herzog et al., 2016).

Plant growth restrictions induced by abiotic stresses like WL can be ameliorated by

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several soil-borne microorganisms that interact positively with plants and that have emerged

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as beneficial tools in modern agricultural systems (Contreras-Cornejo et al., 2016; AbuElsaoud et al., 2017). Among the beneficial microbes, Trichoderma spp. have gained much interest due to their high reproductive capacity and survivability under unfavorable growth

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conditions. Rhizosphere-competent Trichoderma strains benefit the host plants by improving germination; increasing the availability, uptake, and use efficiency of nutrients; and stimulating plant defenses against biotic and abiotic stresses (Kottb et al., 2015; Khaledi and

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Taheri, 2016). Trichoderma-induced growth enhancement in plants under stress is the cumulative result of several factors, including modulations in the phytohormone profile, production of phytoalexins and secondary metabolites, and enhancement of root branching system and nutrient uptake (López-Bucio et al., 2015; Vargas-Inciarte et al., 2019). However, some researchers have reported direct beneficial effects of Trichoderma isolates on several plant growth parameters (Shanmugaiah et al., 2009; Shoresh et al., 2010). For 4

example, in tomato, inoculation with Trichoderma harzianum (TH) promoted plant growth by increasing root growth, inducing secondary root development, and modulating the root system architecture (López-Bucio et al., 2015). Tomato (Solanum lycopersicum) is one of the most important vegetable crop plants consumed all over the world (Heuvelink, 2005). It is grown commonly in tropical, subtropical, and warm temperate climates. Field-grown tomatoes are frequently exposed to unfavorable environmental conditions, such as drought and salinity, and tomato plants can

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experience WL due to excess water caused by heavy rains or cyclones. Current estimates indicate that 16% of the tomato-growing areas worldwide are prone to flooding and WL

(Colmer and Greenway, 2011; Sun et al., 2018). Adverse environmental conditions like WL

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stress have also worsened due to climate change and will be expected to affect the production of crops like a tomato in the coming years. Therefore, a better understanding is needed

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regarding the different physiological, biochemical, and molecular mechanisms that occur in

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tomato during WL stress to allow the design of management techniques that will ensure plant survival and maintain crop yields. The present investigation was designed to evaluate the beneficial effect of a Trichoderma harzianum (KJ831197.1) isolate on tomato plants growing

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under WL stress and to explore the possible protective mechanisms induced by the isolate at the physiological, biochemical, hormonal, and molecular levels.

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2. Materials and Methods

2.1 Experimental material preparation

Trichoderma harzianum was isolated previously from Vicia faba field soil using potato dextrose agar (PDA) medium in Petri plates and genetically typed by DNA sequencing of 5

PCR-amplified ITS1 region of the rRNA gene, which can identify fungi at the species level. The TH isolate was grown and maintained on PDA medium for seven days and then stored at 4oC for further use. Five discs (5 mm diameter) of mycelial agar plugs from seven-day-old growing TH colonies were taken from the PDA plate margin and added to 1 kg of sterilized Zea mays seeds in 1 L flasks. The flasks were incubated at 25 ±2°C for two weeks before mixing with sterile Nile Delta clay soil at a 1:5 ratio. Five sterile discs of PDA medium were inoculated in a control flask to test for any contamination.

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Seeds of tomato (Solanum lycopersicum) were surface sterilized and germinated in peat moss for 35 days, irrigated regularly with H2O, and subsequently transplanted into 15 × 20 cm pots. Treatments included control (C), Trichoderma harzianum (TH), waterlogged

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(WL), and WL+TH. Waterlogging stress was imposed using double-layer plastic bags with a water level 10 cm high in the bottom to facilitate continuous waterlogging and hypoxic

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condition. Water was allowed to enter into the pot by the bottom holes. The plants were

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grown for four weeks in a greenhouse maintained at 23/20°C and 65–70% humidity. The pots were irrigated with 100 mL distilled water every three days and were placed in the greenhouse in a completely randomized block design. The seedlings were analyzed at two

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different growth periods: 14 and 28 days after treatment.

2.2 Estimation of soil pH, N, organic carbon, and organic matter and determination of

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soil bacteria counts

Soil pH was determined as described previously (Reeuwijk, 1995) using a soil-to-water ratio of 1:2.5. Organic carbon (OC) was determined according to the wet digestion method (Walkley and Black, 1934), and organic matter (%) was determined by multiplying the OC

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(%) value by 1.724. Total N was determined by the Kjeldahl method (Lennox and Flanagan, 1982) after digesting the samples in H2SO4.

2.3 Measurement of growth parameters

Plant height was measured using a standard scale, and the numbers of surviving leaves were determined at 14 and 28 DAT. Fresh weights of shoots and roots and crowns were measured

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immediately after uprooting the seedlings, and dry weights were determined after oven drying the samples for 24 h at 70oC.

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2.4 Estimation of photosynthetic pigment content

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Chlorophyll (chl) was analyzed according to the method of Arnon (1949). Fresh tissue was

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extracted in 10 mL 80% (v/v) acetone by grinding with a pestle and mortar. The extract was filtered, centrifuged at 300×g for 20 min, and the supernatant read at 645 and 663 nm. Carotenoids content was measured according to Lichtenthaler (1987).

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For determination of anthocyanin levels, 0.5 g leaf sample was soaked in 3 mL of acidified methanol (1% v/v HCl) for 12 h in darkness at 4°C with occasional shaking. The mixture was then centrifuged for 10 min at 14000×g at 4°C. Absorption of the supernatant

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was read at 530 and 657 nm in a spectrophotometer (Mancinelli, 1984).

2.5 Determination of electrolyte leakage, lipid peroxidation, and hydrogen peroxide

For estimation of lipid peroxidation, 100 mg of fresh leaf tissue was extracted in 1% trichloroacetic acid (TCA), and the extract was centrifuged for ten min at 10,000×g. A 1.0 7

mL sample of the supernatant was mixed with 4 mL of thiobarbituric acid (0.5%, TBA) and heated for 30 min at 95°C. The samples were then cooled in an ice bath and again subjected to centrifugation at 5000×g for 5 min. The absorbance of the supernatant was read at 532 and 600 nm (Heath and Packer, 1968). Hydrogen peroxide (H2O2) was determined by homogenizing 100 mg of fresh leaf tissue in 0.1% TCA. The homogenate was centrifuged at 12,000×g for 15 min. A 0.5 mL sample of supernatant was mixed with 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1

H2O2 was used for calculations (Sergiev et al., 1997).

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mL of potassium iodide (1 M), and the absorbance was read at 390 nm. A standard curve for

The method described by Dionisio-Sese and Tobita (1998) was employed for

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measurement of electrolyte leakage. Pieces of leaves were kept into a test tube containing

deionized water and heated at 40 °C. Therefore, test tubes were cooled at room temperature

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and primary electrical conductivity (EC1) was collected using CON 700 EC meter. Again, the

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test tubes were heated using an autoclave and cooled at room temperature and, thus, final electrical conductivity (EC2) was observed. To calculate EL, the following formula was used:

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EL (%) = EC1/ EC2 × 100.

2.6 Determination of proline, sugar, glycine betaine, and soluble protein contents

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Proline (Pro) content was estimated following the method of Bates et al. (1973). Sugars were extracted and determined according to Irigoyen et al. (1992) using a standard curve of Dglucose for calculation; values were expressed as mg g−1 FW. Total soluble protein was determined following the method of Bradford (1976) using BSA as a standard.

2.7 Total oxidant and antioxidant capacity 8

Total oxidant capacity was estimated according to the method of Erel (2005). Reagent 1 (225 μL) containing 150 μM xylenol orange, 140 mM NaCl, and 1.35 M glycerol in 25 mM H2SO4 (pH 1.75) was added to 35 μL of extract, and the absorbance was read at 560 nm. Subsequently, 11 μL of Reagent 2, containing 5 mM ferrous ion and 10 mM o-dianisidine in 25 mM H2SO4, was added and the absorbance read again at 560 nm after 3 min. For the total oxidant capacity, the results were expressed in terms of micromoles of hydrogen peroxide.

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Total antioxidant activity was determined according to Erel (2004), based on the measurements of the reduction of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid;

ABTS) radical. A 225 μL volume of Reagent 1 containing acetate buffer (pH 5.8) was mixed

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with 5 μL of extract and the absorbance was read at 420 nm. Subsequently, 20 μL of Reagent 2 containing 30 mM ABTS in acetate buffer (pH 3.6) was added and incubated for 5 min, and

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the absorbance was read again at 420 nm. The differences in the absorbance at 420 nm before

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and after the addition of Reagent 2 were considered to represent the total antioxidant activity.

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2.8 Determination of total phenolic and flavonoid contents

Total phenolic content was estimated using the method described by Pinelo et al. (2004), with slight modifications. A 100 μL volume of extract was added to 1.5 mL Folin–Ciocalteu

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reagent solution and incubated at room temperature for 1 min. Subsequently, 1.5 mL of sodium carbonate solution was added and incubated for 90 min in the dark at room temperature. Absorbance was read at 765 nm. Total phenolic content was determined from a calibration curve of gallic acid and expressed as mg g−1 dry weight. The method of Quettier-Deleu et al. (2000) was followed for determination of flavonoids. Dry powdered samples were extracted in ethanol and the extract was centrifuged 9

at 5000×g for 10 min. The supernatant (1 mL) was mixed with 1 mL of 2% aluminum chloride, the mixture was incubated for 10 min at room temperature, and then the absorbance was read at 430 nm and compared to that of catechin standards.

2.9 Assay of sucrose phosphate synthase, pyruvate decarboxylase, and lactate dehydrogenase

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Sucrose phosphate synthase was assayed according to Hubbard et al. (1989) and the sucrose content was determined according to Roe (1934). Pyruvate decarboxylase (PDC) activity was measured according to Morrell et al. (1990). Lactate dehydrogenase (LDH) was measured

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according to Rivoal et al. (1989).

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2.10 Determination of mineral ion contents

For evaluation of N, P, and K contents, dried leaf samples were placed overnight in a muffle furnace at 500°C to make gray ash. After cooling, 10 mL of 6 M HCl was added and the

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samples were boiled to dryness in a water bath, followed by addition of 2 mL HCl. Subsequently, 10 mL double distilled water was added and the sample was filtered through a Millipore filter to remove any residue. The phosphorus (P) content was measured by the

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vanadomolybdate method. Total nitrogen (N) was measured according to McGill and Fiqueiredo (1993), and potassium (K) was estimated by flame photometry.

2.11 Measurement of ethylene and abscisic acid

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For estimation of ethylene concentration, tomato plants were carefully removed from the pots and washed with distilled water. The plants were then kept in conical tubes for 30 min, and then 1 mL samples of evolved gas were withdrawn with a plastic syringe and injected into a GC equipped with a flame ionization detector (Shimadzu GCMS, Japan) and a Shim-pack column using N2 carrier gas (25 mL min−1) (Cristescu et al., 2013). Abscisic acid (ABA) concentrations were determined according to Siciliano et al. (2015). A 500 mg sample of leaf material was extracted in a chilled mortar with 5 mL of 80%

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(v/v) methanol, and the extract was subjected to cold sonication for 30 sec on ice. The samples were then centrifuged at 13,000×g for 5 min at 4°C and the supernatants were

filtered through Whatman filter paper No.1 and analyzed by HPLC–LCMS (Shimadzu

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LCMS-2020, Japan) mass spectrometry. A 20 µL aliquot was injected into an ACE UltraCore

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2.5 SuperC18 column at a flow rate of 0.5 mL min−1.

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2.12 Total RNA extraction and gene expression analyses

Total RNA was extracted from 100 mg fresh leaf tissue using an RNA extraction kit

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(QIAGEN, CA) and following the manufacturer’s instructions. The synthesized cDNA was used for qRT-PCR using SYBR-Green master mix on a Rotor-Gene 6000 instrument (QIAGEN, CA) using the primers listed in Table 1 and actin as an internal control. Gene

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expressions were calculated using the 2-∆∆∆Ct method from three independent experiments (Livak and Schmittgen, 2001).

2.13 Statistical analysis

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The collected data were subjected to multivariate analysis and analysis of variance (ANOVA) using the SPSS version 23 for Mac OS to determine differences among the treatments. The means were compared using Duncan’s Multiple Range Tests (DMRTs) at p<0.05.

3. Results 3.1 Effect of waterlogging and Trichoderma harzianum on soil parameters

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Various soil parameters for the different treatments are presented in Fig. (1A-D). The soil pH for all treatments ranged from 7.73 to 7.80, and the differences in soil pH were not significant among the treatments. Other soil parameters were as follows: soil organic carbon (from 0.79

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to 1.67 %), organic matter (from 1.43 to 2.71), and nitrogen (0.06 to 0.18 %) (Fig. 1A-D).

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Trichoderma harzianum pretreatment

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3.2 Waterlogging-induced declines in growth parameters are ameliorated by

The effect of WL stress on various growth parameters in tomato seedlings are presented in

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Table 2. After 28 days of WL, significant decreases were observed in plant height and numbers of surviving leaves, whereas the number of crowns increased (Table 2).

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Treatment with TH restored the plant height and number of surviving leaves by 20.08 and 18%, respectively, over the WL counterparts (Table 2). Relative to the control, the number of crowns was increased by 28% in the TH-inoculated plants after 28 days of WL (Table 2). Plants subjected to WL showed a decline in biomass accumulation (Fig. 2). After 14 days of WL stress, the whole plant and shoot biomasses decreased by 47 and 55%, respectively. Inoculation of WL plants with TH improved the whole plant and shoot 12

biomasses by 25 and 30% over the WL plants after 28 days after treatment (DAT) (Fig 1). Under normal growth conditions, inoculation with TH increased the accumulation of shoot, root, and whole plant dry mass by 4, 10, and 5%, respectively, after 28 DAT (Fig 2). These effects were maintained under WL conditions and resulted in significant amelioration of the WL-induced decline (Fig. 2).

3.3 Mineral contents increase in Trichoderma harzianum-inoculated plants under

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waterlogging stress

Nitrogen (N), phosphorus (P), and potassium (K) were estimated in plant leaves under

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treatments of WL and TH at different growth periods (Fig. 3). The N concentration was

decreased under WL stress conditions, but treatment with TH improved the N concentration

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in plant leaves by 18% over the WL seedlings after 14 DAT (Fig. 3A). Phosphorus content

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decreased by 62% due to WL versus the control (Fig. 3B), whereas inoculation with TH improved P content by 49% after 14 DAT and this effect was maintained after 28 DAT as well. Potassium levels decreased due to WL stress over the control, but treatment with TH

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improved leaf K content by 60% after 14 DAT. Potassium concentration improved with time

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in both normal as well as TH inoculated plants from 14 to 28 DAT (Fig. 3C)

3.4 TH inoculation improves photosynthetic pigments Photosynthetic pigment contents, including chl a, chl b, total chl, and carotenoids,

exhibited a significant decline under WL conditions. In WL seedlings, leaf chl content decreased by 11% over the control after 14 DAT (Fig. 4A), while chl b declined by 19% due to WL stress (Fig. 4B). Treatments with TH significantly improved the photosynthetic 13

pigment contents (chl a, chl b, and total chl) and also ameliorated the WL induced decline (Fig. 4A-C). Carotenoid content also showed a considerable reduction under WL stress; however, it was significantly improved by inoculation with TH (Fig. 4E). The leaf photosynthetic pigments showed a significant improvement after inoculation with TH (Fig. 4A-E). Leaf sugar content also showed considerable decline under the WL stress, but

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pretreatment with TH significantly improved the sugar content (Fig. 4F).

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3.5 Trichoderma harzianum inoculation enhances the content of secondary metabolites

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TH inoculation improved the contents of Pro, flavonoid, anthocyaninand soluble protein over the control and the WL plants. In response to WL, the proline and total soluble protein contents increased (Fig. 5A-C). TH inoculation improved the anthocyanin content under WL

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conditions. However, these parameters significantly increased by TH pretreatment as

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compared to WL alone (Fig. 5B).

Flavonoid contents were maximal in seedlings exposed to WL and pretreated with TH when compared to the control and to the WL seedlings (Fig. 5C). Flavonoid levels slightly increased in response to WL stress; however, they increased significantly with TH inoculation at 14 and 28 DAT (Fig. 5C). The anthocyanin content was reduced significantly 14

by WL stress, and TH pretreatment ameliorated the decline to a considerable extent. Under normal conditions, TH was effective at enhancing anthocyanin accumulation (Fig. 5D).

3.6 Trichoderma harzianum improves the activities of sucrose synthase, lactate dehydrogenase, and pyruvate decarboxylase

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The activities of sucrose synthase (SuS), lactate dehydrogenase (LDH), and pyruvate decarboxylase (PDC) increased in response to pretreatment of TH when compared to their

activities under WL stress. Leaf SuS activity increased in response to WL and increased was

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imparted due to TH (Fig., 6A). Under control conditions, the levels of LDH activity were low; however, under hypoxic WL conditions, the activity of lactate dehydrogenase

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significantly increased (Fig. 6B). During the transition from 14 to 28 days of growth, the

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LDH activity decreased significantly. Relative to the control, the activity of PDC showed significant increases of 19, 65, and 66% in response to the TH, WL, and WL+TH treatments

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(Fig. 6C). The enzyme activity level decreased slightly with time from 14 to 28 DAT.

3.8 Trichoderma harzianum pretreatment reduces the oxidative damage under

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waterlogging conditions

Pretreatment with TH significantly reduced the generation of hydrogen peroxide and resultant ion leakage. Relative to the control, the content of hydrogen peroxide increased significantly by 27% after 14 days of WL, resulting in a 19% increase in ion leakage (Fig. 7A-B). Pretreatment with TH decreased both the hydrogen peroxide production and the ion leakage.

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However, MDA levels were lower in WL seedlings than in the control (Fig. 7C), and treatment with TH had no marked effect on lipid peroxidation. Total oxidant capacity (TOC) showed 31% increase in WL plants compared to the untreated control, and inoculation with TH decreased the TOC in WL plants (Fig. 7D). By contrast, the total antioxidant capacity (TAC) decreased significantly, by 34 and 19%, in the

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WL and WL+TH treatments, respectively (Fig. 7E)

3.9 Effect of TH pretreatment and WL on ethylene and ABA synthesis

Seedlings pretreated with TH displayed differential regulation of ethylene and ABA

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synthesis under normal and WL stress conditions. Ethylene and ABA levels increased

significantly in WL seedlings over the control. However, ethylene was further increased in

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response to TH, while ABA levels declined. Under normal growth conditions, TH

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pretreatment enhanced both ethylene and ABA levels and the concentrations of ABA were higher than in the WL counterparts. No significant increases in their concentrations were

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noted with growth period (Fig. 8).

3.10 TH pretreatment differentially regulated relative gene expressions

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The relative expression of the ARE gene (are) was increased 1.41-fold, 3.38-fold, and 3.31fold over the control in seedlings subjected to TH, WL, and WL+TH treatments, respectively (Fig. 9A). Inoculation with TH significantly downregulated ARE gene expression in seedlings under WL conditions (Fig. 9A). Under WL stress, ARE gene expression was increased significantly with time (at 2, 4, and 6 DAT).

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The relative expression of the ERFs gene in plants under WL conditions treated with TH is presented in Fig. 9B. The relative gene expression of the ERFs gene was significantly upregulated to 3.47 in the waterlogged seedlings (2 DAT); however, expression decreased slightly to 3.10 after pre-treatment with TH under WL stress (Fig. 9B). Aquaporins (AQPs) are a class of channel-forming proteins that play an essential role in water transport in plants. Analysis of the aquaporin-encoding gene by qPCR revealed upregulation by WL treatment (Fig. 9C). The relative aquaporin gene expression at 2 DAT

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was upregulated to 3.32 and 2.67 in waterlogged seedlings and waterlogged seedlings pretreated with TH, respectively (Fig. 9C). The WL treatment did not induce any significant

changes in aquaporin gene expression with time at 2, 4, and 6 DAT. The inoculation with TH

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slightly decreased aquaporin gene expression; however, the expression increased again at 6 DAT.

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Alcohol dehydrogenase (ADH) gene expression showed no significant changes in

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response to WL stress or treatment with TH. The Fe-superoxide dismutase gene showed a slight increase to 1.61 with WL stress; however, expression decreased to 1.41 in response to inoculation with TH. Pre-treatment of seedlings with TH significantly downregulated genes

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encoding Fe-SOD under WL conditions (Fig. 9D).

The relative expression of the ACO gene showed a significant upregulation to 3.4fold at 2 DAT in response to WL compared to the untreated control. However, treatment with

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TH significantly downregulated the ACO gene expression to 0.99-fold compared to the WL treatment (Fig. 9E). TH treatment showed a significant role in controlling the ACO gene under WL conditions at all growth periods.

4. Discussion 17

The introduction of novel techniques for strengthening indigenously occurring stress tolerance mechanisms in plants can be vital for preventing growth inhibition and productivity losses. The TH pretreatment tested in this study was effective in ameliorating the WLinduced declines in growth parameters of height, fresh weight, and dry biomass. In our study, WL stress had a negative impact on the plant height and the number of surviving leaves, but TH pretreatment ameliorated these responses. The presence of excess soil water negatively

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affects the plant growth phases and alters development and plant survival (Ezin et al., 2010; Kumar et al., 2013).

The growth and development of tomato plants at the two different growth periods

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were reduced under WL stress. Stress-mediated plant growth reduction is generally

manifested as reductions in stem elongation, leaf expansion, and numbers of leaves (Sanchez-

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Rodriguez et al. 2010). In the present study, tomato seedlings tended to adapt to flooding

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through increased adventitious root formation (Chen et al., 2016; Song et al., 2011) or through the regulation of ion uptake (Colmer and Flowers, 2008; Colmer and Greenway, 2011).

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Application of TH mitigated the deleterious impacts of WL stress and considerably stimulated the growth of tomato seedlings. Trichoderma spp. promote root development and the formation of secondary roots by stimulating phytohormone production that includes

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auxins, gibberellins, and cytokinins, as reflected in increased leaf area and stress amelioration (Hashem et al., 2014). TH could increase nutrient uptake and root colonization, thereby physiologically enhancing the availability of P and Fe to plants and resulting in significant increases in dry weight, shoot length, and leaf area (Owen et al., 2015). Under WL stress, soil oxygen status may be either hypoxic (low) or anoxic (absent) and this inhibits plant growth by impairing photosynthetic activities by inhibiting the 18

synthesis of pigments and net photosynthetic rates (Voesenek and Bailey-Serres, 2013, 2015). In the present study, TH pretreatment protected the seedlings from WL-mediated declines in photosynthetic pigment synthesis. Ahmad et al. (2015) previously demonstrated increases in chlorophyll synthesis in Brassica juncea. The increased growth in TH pretreated seedlings in the present study under normal and WL stress can be attributed to a higher uptake of mineral ions, including N, P, and K. Mineral macroelements regulate critical physiological and biochemical processes, including

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cell division, enzyme activity, and synthesis of proteins like Rubisco (Ahanger and Agarwal, 2017; Hasanuzzaman et al. 2018; Ahanger and Ahmad, 2019). In the present study, the

integrity of membranes was significantly disrupted due to WL effects on ion uptake and

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concomitant WL-induced reductions in ATP availability due to effects on photosynthesis. ATP is required for the optimal functioning of membrane pumps and decreases ATP

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production may have potentially contributed to the reduced mineral ion uptake (Lei et al.,

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2012; Ren et al., 2016). The degree of membrane protection from the WL-induced stress effects can be linked to protective molecules, like osmoprotectants (e.g., sugars and amino acids) (Ren et al., 2016), and to the antioxidant defense system (Ahanger et al., 2017), which

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maintain membrane functioning and protect the ion exchange capacity of the plasma membrane.

The maintenance of membrane stability is an essential requisite for sustaining the

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physiological functioning of cells, and parameters related to the membrane stability index (MSI) have been considered as indicators of stress tolerance (Chen et al., 2013; Zhou et al., 2016). In the present study, a significant decline in the generation of H2O2 was observed in response to the TH pretreatment, as reflected by a lower ion leakage, and this positive impact was maintained under WL conditions. Stress-mediated generation of excessive H2O2 levels induces results in the lipid peroxidation responsible for the adverse effects on membrane 19

function (Lei et al., 2012; Hasanuzzaman et al. 2013; Bansal and Srivastava, 2015). Our results show a remarkable increase of MDA accumulation in the untreated seedlings than in the TH-pretreated seedlings. The reductions in MDA levels in the TH-pretreated tomato seedlings might reflect an elevated expression of enzymes and the synthesis of compounds involved in eliminating the toxic molecules associated with lipid peroxidation, including ROS (Parida and Das 2005; Ahmad et al. 2015). The data presented in the current study show that TH has the potential to enhance the expression of antioxidant enzymes and other

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protective molecules, with the cumulative effect of improving redox homeostasis and eliminating the toxic free radicals responsible for peroxidation of lipids.

Previous studies have shown that the production of sufficiently high sugar levels is a

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crucial factor in plant tolerance to long-term submergence (Perata et al., 2011). In the present study, the soluble sugar content was decreased. This indicated that the intolerant tomato

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seedlings consumed large amounts of soluble sugars during WL stress. Increased

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accumulation of osmolytes helps stress-exposed plants to maintain their cellular functions by protecting the major metabolic processes (Hasanuzzaman et al. 2019). Increased Pro accumulation under WL stress has been reported to prevent photosynthetic inhibition by

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improving water use efficiency (Ou et al., 2011; Yang et al., 2014). In the present study, TH promoted the accumulation of Pro which may have assisted tomato seedlings in avoiding the WL-induced photosynthetic and metabolic arrests to a considerable extent.

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TH pretreatment resulted in differential regulation of the synthesis of ethylene and ABA in tomato seedlings. WL stress triggered the accumulation of ethylene and ABA to maintain the stress-mediated generation of these hormones needed for initiating the downstream signaling events for stress mitigation. However, TH pretreatment maintained the phytohormone concentrations at levels below those that would trigger stress-induced generation of ethylene and ABA. This avoided activation of the downstream events and 20

allowed a quick stress response in tomato. Increased ethylene production in response to water stress has been demonstrated by Voesenek and Bailey-Serres (2015). However, reports discussing the role of TH in the regulation of WL tolerance through modulations in phytohormone profile are lacking. In the present study, TH pretreatment tended to maintain sufficient endogenous levels of ethylene and ABA to initiate the tolerance mechanism and prevent the arrest of key processes like photosynthesis and redox homeostasis. Importantly, the overcoming of oxygen-deprived conditions by the formation of lysigenous aerenchyma

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and adventitious roots has been correlated with ethylene production stimulated by ROS production (Steffens and Rasmussen, 2016; Yamauchi et al., 2017). Overproduction of

ethylene-responsive proteins has been reported to regulate several growth, developmental,

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and physiological events, including cell expansion and leaf formation, through overexpression of genes like ACS8 (Plett et al., 2014), EIN2 (Feng et al., 2015), BOLITA

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(Marsch-Martinez et al., 2006), ERF6 (Dubois et al., 2013), and multiple other ERF-encoding

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genes. ABA negatively regulates adventitious root emergence in an antagonistic interaction with ethylene (Benschop et al., 2005). Therefore, our study suggests that interactions occurring between ethylene, ABA, and ROS promote flooding stress response in TH-

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pretreated plants.

Phenolic compounds, including flavonoids, tannins, and other related secondary metabolites, strengthen the non-enzymatic antioxidant system and favor elimination of ROS

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and prevention of lipid peroxidation by altering the peroxidation kinetics, thereby maintaining membrane fluidity (Ahmad et al.2008, 2010; Jin et al., 2016; Choudhury et al., 2017). Flavonoids protrude into the lipid membranes and prevent the diffusion of free radicals, thereby inhibiting their toxicity at other cellular locations. The expression of different genes revealed differential patterns for Fe-SOD and ADH, which increased with TH treatment, and for ERF, ACO, ARE, and aquaporin, which 21

decreased. The expression of SOD and ADH was upregulated in TH pretreated seedlings under normal as well as WL conditions, indicating a beneficial role of TH in ROS elimination and sugar metabolism. SOD is a key ROS-neutralizing enzyme that prevents the accumulation of superoxide and disruption of photosynthesis (Ahanger and Agarwal, 2017; Ahmad et al., 2015). During WL, the enzymes of the fermentation pathway, including ADH, pyruvate decarboxylase (PDC), and sucrose synthase (SUS), are upregulated to maintain sugar metabolism (Ismond et al., 2003). The expression of PDC and ADH is regulated by the

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ethylene-responsive factors HRE1 and HRE2 (Hess et al., 2011). Aquaporins are exclusively localized on the tonoplast and plasma membranes, and the regulation of aquaporin gene

expression and activity is a vital component of tolerance to stress conditions and depends

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on complex processes and signaling pathways (Kapilan et al., 2018). In the present study, the TH-mediated reduction in aquaporin gene expression may have contributed to the

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prevention of influx of extra water into the plant tissues, thereby protecting metabolism.

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The growth of tomato seedlings was significantly affected by WL stress, and TH pretreatment was significantly beneficial in preventing WL-associated growth suppression. TH pretreatment reduced WL-induced oxidative damage, protected membrane permeability,

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and improved mineral uptake, thereby leading to better growth and photosynthetic performance. Reductions in oxidant status and increased antioxidant status due to TH can be attributed to the greater synthesis of metabolites and redox components. Enhanced ethylene

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and ABA production mediated by TH treatment allowed the quick generation of signaling events for the amelioration of WL-induced damage by triggering stress tolerance mechanisms. Further studies focusing on the identification of the relevant signaling components will be interesting. In this respect, the expression profiling in the present study has identified a number of specific genes that affect antioxidant, ethylene, and sugar metabolism and transport processes in WL-stressed tomato plants (Fig. 10). 22

Author Statement A.A.E., and M.H. designed the experiment. A.A.E. S.H.Q. and M.H.S. conducted the experiment. H.A.S.A. provided the reagents and wrote the manuscript draft. A.A.E. and M.H. analyzed the data. A.A.E., H.A.S.A, S.H.Q., M.H.S. wrote the manuscript draft. M.H. critically reviewed and edited the manuscript. All authors approved the final manuscript and

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agreed for submission.

Conflict of Interest statement

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The authors declare no conflict of interest.

Acknowledgement

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We thank Khursheda Parvin Hira and Sayed Mohammadd Mohsin, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh and M. H. M. Borhannuddin

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Bhuyan, Bangladesh Agricultural Research Institute, Jaintapur, Sylhet for their critical

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reading and formatting of the manuscript.

23

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Shoresh, M., Harman, G.E., Mastouri, F., 2010. Induced systemic resistance and plant responses to fungal biocontrol agents. Annu. Rev. Phytopathol. 48, 21–43. doi:10.1146/annurev-phyto-073009-114450 Siciliano, I., Amaral Carneiro, G., Spadaro, D., Garibaldi, A., Gullino, M.L., 2015. Jasmonic acid, abscisic acid, and salicylic acid are involved in the phytoalexin responses of rice to Fusarium fujikuroi , a high gibberellin producer pathogen. J. Agric. Food Chem. 63, 8134–8142. doi:10.1021/acs.jafc.5b03018

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Song, J., Shi, W., Liu, R., Xu, Y., Sui, N., Zhou, J., Feng, G., 2017. The role of the seed coat in adaptation of dimorphic seeds of the euhalophyte Suaeda salsa to salinity. Plant

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Steffens, B., Rasmussen, A., 2016. The physiology of adventitious roots. Plant Physiol. 170, 603–617. doi:10.1104/pp.15.01360

Sun, Q., Wang, Y., Chen, G., Yang, H., Du, T., 2018. Water use efficiency was improved at

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against Fusarium oxysporum wilt in tropical greenhouses. Agron. Costarricense 43, 85-100. Doi:10.15517/rac.v43i1.35671 Voesenek, L. a. C.J., Bailey-Serres, J., 2013. Flooding tolerance: O2 sensing and survival strategies. Curr. Opin. Plant Biol. 16, 647–653. doi:10.1016/j.pbi.2013.06.008 Voesenek, L.A.C.J., Bailey-Serres, J., 2015. Flood adaptive traits and processes: an overview. New Phytol. 206, 57–73. doi:10.1111/nph.13209 Walkley, A., Black, I.A., 1934. An examination of the degtjareff method for determining soil

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flooding when growing in species-rich plant communities. New Phytol. 213, 645–

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90. doi:10.1080/11263504.2014.976294

36

14 Days a er treatment 3

(A)d

28 Days a er treatment

(B)

ANOVA: F-ra o=203.6; p<0.001*

d

2.5

b

2

b

b

a

c

b

ab

a

a

ab

ab

7.7

a

a

1.5

a

Soil pH

Soil nitrogen (%)

ANOVA:F-ra o=1.9; p=0.136n.s.

8.1

7.3

1 6.9

0.5 6.5

0

f

g e

Waterlogging

ANOVA:F-ra

Trichoderma +Waterlogging o= 141.4; p<0.001*

3

ef d

c

1

b

a

0.8

d

2

b

0.4

0

0

Waterlogging

Control

Trichoderma +Waterlogging

c

b

Trichoderma

Waterlogging

a

Trichoderma +Waterlogging

re

Trichoderma

-p

0.5 0.2

b

a

1.5

1

0.6

Control

Trichoderma Waterlogging Trichoderma +Waterlogging ANOVA:F-ra o=110.3; p<0.001*

2.5

1.4 1.2

Control

(D)d

ro of

Soil organic carbon (%)

1.6

(C)

Trichoderma

Organic ma er (%)

1.8

Control

Treatments

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Figure 1. Various soil parameters for different treatments, including the control, inoculation with Trichoderma harzianum (TH), waterlogging (WL), and interaction between TH and WL

ur na

waterlogging. Statistical differences among the samples are labelled with different letters

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according to Duncan’s multiple range tests.

37

ro of -p re

lP

Figure 2. Inoculation with Trichoderma harzianum (TH) restored plant biomass in terms of fresh weight (g FW plant-1) and dry weight (g DW plant-1) for shoot, roots, and whole plants

ur na

after exposure to waterlogging (WL) stress. Statistical differences among the samples are

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labeled with different letters according to Duncan’s multiple range tests.

38

3

(A)

N content (mmol g-1 DW)

2.5

2

f d

g

ANOVA: F-ra o= 140.1 p<0.001*

e

14 Days a er treatment

a

1.5

b

a

c 28 Days a er treatment

1

0.5

Trichoderma Waterlogging ANOVA: Treatments

F-ra o= 128.2; p<0.001*

h

1.2

e

d c b

0.4

a

d

1.2

c 0.8

b

ANOVA: F-ra o= 456.6; p<0.001*

b

a

0.4

0

0

Trichoderma Waterlogging Trichoderma +Waterlogging

a

a

Control

Trichoderma Waterlogging Trichoderma +Waterlogging

re

Control

e

(C)

1.6

g f

0.8

2

Trichoderma +Waterlogging

ro of

Control

-p

(B)

K content (mmol g-1 DW)

P content (mmol g-1 DW)

0 1.6

Treatments

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Figure 3. Mineral nutrition in terms of nitrogen (N), phosphorus (P), and potassium (K) (mmol g−1 DW) in leaves under different treatments, including the control, inoculation with

ur na

Trichoderma harzianum, waterlogging (WL), and interaction between TH and WL. Statistical differences among the samples are labeled with different letters according to Duncan’s

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multiple range tests.

39

Waterlogging

Control

28 Days a er treatment

14 Days a er treatment

d

ANOVA: F-ra o=460.0; p<0.001*

e d

4

d

(B) d

e

e

ANOVA:F-ra o=366.1; p<0.001*

f

c

44

c

c

42

b a

40

b

3

2

38 1 36

d

Waterlogging

(D)Control

de

e

14

f b

c a

Trichoderma

Waterlogging

13

Chl-a/b

30

d

b

12

lP

20

a

Trichoderma

ANOVA: F-ra o=109.0; p<0.001* +Waterlogging

Treatments

d

c

40

a

c

c

a

11

10

10

0

0.3

0.2

Control

(E)

e

Trichoderma

f

Waterlogging

ANOVA: F-ra Treatments

Trichoderma

e

d

c

b

a

Jo

0.1

Trichoderma

Control

(F)

Trichoderma

Waterlogging

ANOVA: F-ra Treatments

g

f

Trichoderma

+Waterlogging o=270.4; p<0.001*

e 12

c

d

d a

b

11

a

0

Control

13

+Waterlogging o=203.6; p<0.001*

ur na

0.4

Caroteinoids (µg g-1 FW)

Trichoderma

ANOVA:F-ra o=507.8; p<0.001* +Waterlogging

Treatments

-p

Trichoderma

Total Sugar contents

Total Chlorophyll (µg g-1 FW)

50

0

Control

(C)

re

34 60

c

a

ro of

Chl a (µg g-1 FW)

46

(A)

Chl-b (µg g-1 FW)

48

Waterlogging+T.harzianum

T. harzianum

10

Waterlogging

Trichoderma +Waterlogging

Control

Trichoderma

Waterlogging

Trichoderma +Waterlogging

Treatments

Figure 4. Photosynthetic pigments (chl a, chl b, and carotenoids) and total sugars content in plants under different treatments. Statistical differences among the samples are labeled with different letters according to Duncan’s multiple range tests. 40

8

9

a

b

c

c

f d

g

e

8

7

6

Control

(C)

6

Trichoderma

c

5

a

Waterlogging

ANOVA: Treatments F-ra o= 456.6; p<0.001*

a

e

d

c b

b

4 3 2 1 0

7

f

d

c

e

b

a

5

4

3

Control

(D)

0.6

d

Trichoderma Waterlogging f ANOVA: Treatments

e

0.7

de

c

a

0.3 0.2 0.1

Control

Trichoderma Waterlogging Trichoderma +Waterlogging

re

v

F-ra o= 108.3; p<0.001*

b

0.4

Trichoderma Waterlogging Trichoderma +Waterlogging

Trichoderma +Waterlogging

c

0.5

0

Control

ANOVA: F-ra o= 444.39; p<0.001*

h

g

6

2 0.8

Trichoderma +Waterlogging

Anthocyanin content (Unit g-1 FW)

Total flao noids (mgru n 100g-1 DW)

5 7

(B)

ro of

ANOVA: F-ra o= 154.2; p<0.001*

Total Soluble proteins (mg g-1 FW)

Proline content (μg g-1 FW)

(A)

28 Days a er treatment

-p

14 Days a er treatment 10

Treatments

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Figure 5. Proline, total soluble proteins, total flavonoids, and anthocyanin contents in plants under different treatments, including control, inoculation with Trichoderma harzianum,

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waterlogging, and their combination. Statistical differences among the samples are labeled

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with different letters according to Duncan’s multiple range treatments.

41

Sucrose Synthase (µmole g-1 FW h-1)

300

(A)

ANOVA: F-ra o=302.4; p<0.001*

e

e d

e

250

200

14 Days a er treatment

ab

a

c

28 Days a er treatment

bc

150

100

50

Trichoderma

Waterlogging

ANOVA: Treatments F-ra o=329.5; p<0.001*

7

Trichoderma +Waterlogging

e

d 6 5 4 3

c

b 2

a

a

a

ab

1 0

0.8

(C)

ANOVA: F-ra o=161.4; p<0.001*

0.7

Trichoderma

Waterlogging

d c

0.6 0.5 0.4

b a

0.3

b

a

0.2 0.1 0

Control

e

de

Trichoderma +Waterlogging

Control

Treatments

Trichoderma

ro of

Control

Waterlogging

Trichoderma +Waterlogging

-p

(B)

Pyruvate decarboxylase (mg-protein min-1)

Lactate dehydrogenase (µmole g-1 FW min-1)

0 8

re

Figure 6. Inoculation with Trichoderma harzianum recovered and enhanced various plant metabolic enzymes: (A) sucrose synthase (B) lactate dehydrogenase (C) pyruvate

lP

decarboxylase, exposed to waterlogging stress. Statistical differences among the samples are

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ur na

labelled with different letters according to Duncan’s multiple range tests.

42

14 Days a er treatment

28 Days a er treatment

16 14

(A) ANOVA: F-ra o= 658.0; p<0.001***

WL

CK

d

Membrane Ion leakage (%)

c 12

b

b

10

a

b

a

a

8

ro of

6 4 2

0 100

W+T

T. Control

(B)

Trichoderma

Waterlogging

Trichoderma 16 +Waterlogging Treatments

ANOVA: F-ra o= 659.8; p<0.001***

(C)

ANOVA: F-ra o= 928.4; p<0.001***

f

80

g

c b

40

d

a

f

d

h

c

b

a

8

re

c

d

e

-p

60

MDA (nmol g-1 FW)

H2O2 (nmol g-1 FW)

12

e

4

TOC (%)

0.4 0.3 0.2

b

f

e

Trichoderma +Waterlogging

80

Trichoderma

Waterlogging

Control

Trichoderma

Waterlogging

Treatments

Trichoderma +Waterlogging

g

h e

f d

b

0

(E)

ANOVA: F-ra o= 1930.2; p<0.001*

d

a

Control

0 100

c

c

Jo

0.1

Waterlogging

ANOVA: F-ra o=99.8; p<0.001***

0.6 0.5

Trichoderma

TAC (%)

0.7

(D)Control

ur na

0 0.8

lP

20

60

c

b a

40

20

0

Control

Trichoderma +Waterlogging

Trichoderma

Waterlogging

Trichoderma +Waterlogging

Treatments

Figure 7. Inoculation with Trichoderma harzianum enhanced antioxidant activities and decreased oxidative damage in plants exposed to waterlogging treatments. (A) Membrane ion leakage (%), (B) H2O2 levels (nmole g−1 FW), (C) Lipid peroxidation (MDA, nmole g−1 FW), 43

(D) Total oxidant capacity (%), (E) Total antioxidant capacity (%). Statistical differences among the samples are labeled with different letters according to Duncan’s multiple range tests.

2

14 DAT (A)

28 DAT

1.8

f

1.6 1.4

e

d

1.2 1

b

0.8

c

ro of

Ethylene (nmole h-1 g-1 FW)

g

ANOVA: F-ra o=326.25; p<0.001*

b

a

0.6 0.4 0.2

Control Trichoderma Waterlogging ANOVA: F-ra o=736.2; p<0.001* Treatments

g

d

500

e c

450 400

a

350

200

ur na

300 250

c

re

f

Trichoderma +Waterlogging

lP

ABA (pmole h-1 g-1 DW)

550

(B)

-p

0 600

Control

Trichoderma

Waterlogging

Treatments

b

Trichoderma +Waterlogging

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Figure 8. Effect of inoculation with Trichoderma harzianum and waterlogging treatments on levels of the plant hormones (A) ethylene and (B) abscisic acid (ABA). Differences among the samples are labeled with different letters according to Duncan’s multiple range tests.

44

14 Days a er treatment

28 Days a er treatment

4.5

d

3.5

3.5

cd b

3

bc

2.5 2

a a a

1

Trichoderma +Waterlogging

e e e

a ab

b a a a

1

b ab

a ab

b

1

Control

Trichoderma

Trichoderma

Waterlogging

cde

cde 1.2

ab abc

0.4

e

e

e

cd

d

bc ab

cd

a

ur na

1

Control

Control

Trichoderma

e

3

lP

e

2.5

bcde abcd

Waterlogging

ANOVA: F-ra o=164.75; p<0.001*Treatments f

3.5

(D)

a

0.6

4

Trichoderma +Waterlogging

f e

de

0.8

4.5

(E)

e de

1

f

f

2

Trichoderma +Waterlogging

Rela ve Expression of ACO gene

Control

ANOVA: F-ra o=46.25; p<0.001*Treatments

Waterlogging

ANOVA: F-ra o=177.3; p<0.001*Treatments

0

0

Rela ve Expression of Fe-SOD gene

1.5

0.2

0.5

0

c

2

1.4

e c

2

0.5

d

2.5

1.6

(C)

ANOVA: Treatments F-ra o=177.3; p<0.001*

2.5

1.5

e

3

ro of

Waterlogging

Rela ve Expression of ADH gene

Rela ve Expression of Aquaporine gene

Trichoderma

3

2.5

f

0 Control

d

1.5

(B)

f

-p

0

3.5

f

f

0.5

0.5

4

ANOVA: F-ra o=329.5; p<0.001*

re

1.5

a a a

4

(A)

e e

Rela ve Expression of ERFs gene

Rela ve Expression of ARE gene

4

ANOVA: F-ra o=329.5; p<0.001*

c

d

Trichoderma +Waterlogging

(F)

f f e

de

c

2

b

1.5 1

a

a

0.5 0

Trichoderma Waterlogging Trichoderma +Waterlogging

Control

Trichoderma Waterlogging Trichoderma +Waterlogging

Treatments

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Figure 9. Trichoderma harzianum-induced changes in the relative expression of various genes in plants exposed to waterlogging treatments. (A) ARE gene, (B) ERFs gene, (C) Aquaporin gene, (D) alcohol dehydrogenase (ADH) gene, (E) Fe-SOD gene (F) ACO gene. Statistical differences among the samples are labelled with different letters according to Duncan’s multiple range tests. 45

Increased secondary Increased activities of Pretreatment metabolites (flavonoid, soluble with TH metabolic enzymes sugar and proline) Enhanced expression of antioxidant Increased enzymes (e.g. Fe-SOD) antioxidant defense Decreased oxidative stress (Decreased EL, MDA and H2O2)

Enhanced photosynthesis pigment contents

Increased plant height, number of leaves and biomass

Enhanced plant N, P and K content

Improved root growth

Improved nutrient uptake

Improved nutrient availability

re

-p

Higher soil organic matter, soil N and C Maintainance of better soil pH

ro of

Hormonal regulatiion (Ethylene and ABA)

Pretreatment with TH

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Fig. 10. Possible mechanisms of Trichoderma harzianum (TH), waterlogged (WL) tolerance

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ur na

in tomato based on the present experiment.

46

Table 1.The sequences of the primers used in qRT-PCR Primer name

Primer sequence (5'–3')

Annealing temperature (°C)

Actin

F: AATGATCGGAATGGAAGCTG

64

R: ATCCTCCGATCCAGACACTG ADH

F: CCTCGTTCGGATATTCCTTG

63

R: GTTTAGTCCGCCATGGTGAT SOD

F: CAACGCTGCTCAGGCGTGGA

67

ARE

F: GAATCGCTGGTGCTTCTAGG R: CTCAGCGATCACCTGTTGAA

ERGs

F: GAAGAGGAGGAGGATGGTGAT R: CAGTTAAGCCACTCTTATCTATC F: AGCCAATCAACTTCCAAACACC

53

64

67

-p

ACO

ro of

R: GGCGGCTCCAAGTCTGGCAC

R: AGTCTACTGTAACTCCTGGTGCC F: GTTCCTATCCTTGCCCCACT

re

Aquaporin

Jo

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lP

R: AGGCGTGATCCCTGTTGTAG

47

67

Table 2. Plant height (cm), number of surviving leaves, and number of crowns in control plants versus plants exposed to waterlogging (WL) or treated with Trichoderma harzianum (TH). Statistical differences among the samples are labeled with different letters according to Duncan’s multiple range tests. Plant height

Number of

Number of

(cm)

surviving leaves

crowns

14

29.80 ± 0.13 c

6.00 ± 0.58c

72.00 ± 4.36a

28

41.73 ± 0.37 e

8.00 ± 0.58c

90.00 ± 3.06b

14

37.97 ± 0.42 d

7.67 ± 0.33b

96.67 ± 4.10b

28

56.37 ± 0.23 f

10.33 ± 0.33a

124.33 ± 4.63c

14

19.07 ± 0.20 a

4.67 ± 0.33e

117.33 ± 2.03c

28

29.33 ± 0.64 c

6.00 ± 0.58f

128.67 ± 2.73c

Trichoderma+waterlo 14

25.43 ± 0.23 b

5.67 ± 0.33d

154.67 ± 4.67d

gging (TH+WL)

36.70 ± 0.32 d

7.33 ± 0.33d

196.00 ± 5.69e

16.07

93.17

Control (CK)

Trichoderma (TH)

Waterlogging (WL)

28

ro of

DAT

-p

Treatment

F-ratio

349.53

ANOVA

p-value

p<0.001 ***

p<0.001 ***

p<0.001 ***

349.53***

16.07***

93.17***

236.41***

13.79***

109.18***

897.71***

37.78***

73.13***

14.29***

0.357 NS

7.513**

re

One-way

F-(THTrichoderma tr.) F-(time)

lP

F-(corrected model)

ur na

F-(WLwaterlogging x THTrichoderma)

NS: not significant (p>0.05); * significant at p<0.05; ** highly significant at p<0.01; ***

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very highly significant at p<0.001; DAT- Days after treatment

48