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Article

Effect of Using Trichoderma spp. on Turfgrass Quality under Different Levels of Salinity

by
Nour S. Abu-Shanab
1,
Kholoud M. Alananbeh
2,
Yahia A. Othman
1 and
Malik G. Al-Ajlouni
1,*
1
Department of Horticulture and Crop Sciences, School of Agriculture, University of Jordan, Amman 11942, Jordan
2
Department of Plant Pathology, School of Agriculture, University of Jordan, Amman 11942, Jordan
*
Author to whom correspondence should be addressed.
Water 2022, 14(23), 3943; https://doi.org/10.3390/w14233943
Submission received: 11 November 2022 / Revised: 27 November 2022 / Accepted: 28 November 2022 / Published: 4 December 2022
(This article belongs to the Special Issue Desalination Treatment of Irrigation Water)
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Abstract

:
Lawns achieve environmental, functional, and aesthetical roles in urban environments. The objectives of this research were to assess the effect of different salinity levels on Trichoderma isolates and to study the effect of Trichoderma spp. on perennial ryegrass under different levels of salinity. T. harzianum (ThLem2017-01) and T. atroviride (TaDP2019-01) isolates had a higher mycelium growth rate than T. atroviride (TaDP2019-02) when salinity levels were low. In contrast, the mycelium growth rate of T. atroviride (TaDP2019-02) isolate at high salinity levels had superior results. Turfgrass seeds that were inoculated with (TaDP2019-02) isolate maintained high radicle length, coleoptile length, and leaf length under high salinity levels. Increasing salinity level decreased clippings’ fresh weight (FW), dry weight (DW), and shoot and root dry weight of perennial ryegrass. Interestingly, perennial ryegrass pots that were treated with (TaDP2019-02) isolate had increased FW and DW by 16 to 114% and 24 to 76%, respectively. Soils that were inoculated with Trichoderma (TaDP2019-02) had higher CO2 respiration (75%) than the control. Therefore, using T. atroviride (TaDP2019-02) isolate revealed promising results in increasing plant biomass and as an environmentally friendly alternative factor to overcome salinity stress.

1. Introduction

Turfgrasses are one of the main components of the landscape [1]. Turfgrasses play an essential role in urban environments by reducing soil erosion, filtering air and water and consequently reducing pollution [2,3]. In addition, lawns add an aesthetic value to the landscape and increase a property’s value [4].
Turfgrasses are used to enhance the space of beauty [5], recreation, and sport [1]. It is used as a playground because it gives a cushioning influence that decreases pains to players when compared to ordinary soils or artificial grass, particularly in contact sports such as rugby and football [3]. Turfgrass is considered as billion-dollar industry [6,7]. Perennial ryegrass (Lolium perenne L.) is one of the most common cool-season turfgrasses. It is native to temperate Asia, Europe, and North Africa [8]. It is used as a forage species with high yield and quality, and is appropriate for parks, home courtyards, cemeteries, golf courses, and roadsides [9]. Perennial ryegrass is often used for quickly re-establishing devastated lawns because it is distinguished by rapid seed germination, growth, and establishment [3]. Interestingly, perennial ryegrass has been used widely for clean-up of heavy metal-polluted soils (remediation) [10].
Turfgrass is affected by numerous abiotic and biotic stresses such as salinity, intensive fertilization schemes, heat, cold, drought, soil compaction, diseases, pests, and pesticide and herbicide application [11,12]. Soil salinity is consistently considered as one of the major problems around the globe. High salt levels in soil ECe values of ˂1.0–24.0 dS m−1 significantly decrease plant performance and quality [13]. However, seed germination and the seedling stage are the most sensitive phases to saline water [14]. In fact, at early growth stages (germination and seedling) salt stress can induce critical osmotic potential that inhibits the water uptake and mineral nutrients of the germinated seeds or/and increase assimilation of toxic ions (especially Na) that possibly alter hormonal or enzymatic activities of the plants [15,16].
Jordan is among the poorest countries in terms of water resources worldwide, facing serious problems related to water shortages [17]. High demand for irrigation water coupled with a significant rise in population recently due to refugees fleeing from the Syrian civil war increase the over-abstraction of groundwater (the main source of fresh water in Jordan) and consequently reduce water quality [18]. In fact, the continuous over-abstraction of water reduced the levels of groundwater and increased the salinity readings [19]. To overcome these problems, it is imperative to modulate some horticultural practices through decreasing the application of chemical fertilizers, proper leaching and drainage, and deep plowing [12,20].
Perennial ryegrass is classified as a cool-season turfgrass, with moderate irrigation requirements, good traffic tolerance, fine texture leaf type, and moderate tolerance to salinity [3,21]. The salinity thresholds of perennial ryegrass ranged from 4 to 8 dS m−1 [22]. This tolerance in perennial ryegrass is partially attributed to antioxidant genes expression efficiency as well as antioxidant enzymes activation [9]. Plant growth (shoot and root biomass, density) and aesthetic visual component (leaf greenness or color) are critical variables to assess turf grass responses to salt stress [23,24]. Growing perennial ryegrass in a saline environment (6 dS m−1) for six weeks significantly reduced shoot and root dry weight and visual quality [24].
Trichoderma spp. Fungi inoculation has been recommended as an environmentally friendly approach to overcome salinity problems [25]. These fungi are used to enhance nutrient uptake and increase plant growth, induce systemic resistance and as a biological control for many plant’s fungal pathogens and pests [26,27,28,29]. Trichoderma symbioses with wheat plants, which ameliorates the negative impact of salt stress by inducing the shoots to accumulate more protective amino acids [30]. In addition, Trichoderma spp. decreases the need for chemical fertilizers and pesticides. In this context, the positive interaction between fungi and plants can achieve the global trend toward sustainable agriculture, by promoting salinity tolerance and lowering agrichemical inputs [18,31]. Therefore, T. harzianum fungi inoculation can be a promising practice for lawn management in saline soils. However, the effect of salt stress on T. harzianum is not well investigated. In addition, no study we are aware of has discussed the influence of T. atroviride inoculation on perennial ryegrass growth and physiology under saline conditions. The objectives of this study were (1) to evaluate the effect of different salt stress levels on Trichoderma isolates growth and (2) to assess the influence Trichoderma spp. inoculation on plant growth and physiology of perennial ryegrass under saline conditions.

2. Materials and Methods

2.1. Experiments Layout

In this research, laboratory and greenhouse experiments were conducted to evaluate the effect of Trichoderma sp. on perennial ryegrass (Lolium perenne L.) under salinity stress. Two experiments were conducted in the laboratory while the third one was in the greenhouse. In the first experiment, the mycelium growth rate of three Trichoderma isolates (Table 1) under different sodium chloride (NaCl) concentrations was assessed. In the second experiment, the performance of perennial ryegrass seeds inoculated with Trichoderma spp. isolates was assessed under saline conditions. In the third experiment, the best Trichoderma isolate from experiment two was used to inoculate perennial ryegrass and evaluated in the greenhouse under a saline environment.

2.2. Trichoderma spp. Isolates

Three Trichoderma isolates belonging to two species, isolated from two plants from the Jordan Valley, Jordan, were used in this study. Isolates TaDP2019-01 and TaDP2019-02 were isolated in 2019 from date palm fronds and identified as T. atroviride; isolate ThLem2017-01 was isolated in 2017 from lemon leaves and identified as T. harzianum. The isolates were molecularly identified by sequencing the internal transcribed spacer (ITS1/ITS4) [32]. Deoxyribonucleic acid (DNA) extraction, polymerase chain reaction (PCR) amplification, and sequencing were conducted at Macrogen Inc, Seoul, South Korea. Sequences were received as FASTA files (stands for “FAST-All”), edited via MEGA 7 software (Institute for Genomics and Evolutionary Medicine, Temple University, Philadelphia, PA, USA) [33] and BLASTn program (National Center for Biotechnology Information, Bethesda, MA, USA). Reference accessions with high similarity were used for phylogenetic analysis. Final sequences with their identity were submitted to the Genbank and accession numbers were received for ITS1/4 (MT626715, MT626716, and MT626718).

2.3. Experiment 1: Mycelial Growth Rate of the Three Trichoderma spp. Isolates under Different Salt Concentrations

Trichoderma spp. isolates (TaDP2019-01, TaDP2019-02, ThLem2017-01) (Table 1) were sub-cultured on potato dextrose agar (PDA) (TCHNO PHARMCHEM, Delhi, India) medium (39.3 g L−1 water (sterile-distilled water) amended with different NaCl concentrations (50, 100, 150, 200, 300, 400, 500, 750, and 1000 mM) (they are equivalent to 4, 8, 12, 18, 28, 38, 44, 64, and 84 dS m−1) by using sodium chloride extra pure (Scharlau), Sentmenat, Spain. Sterile-distilled water was used as a control. Media were sterilized by autoclaving at 121 °C and 15 psi then poured into 9 cm diameter Petri dishes under aseptic conditions [34].
One disc (2 mm in diameter) from 10-day-old cultures of each Trichoderma spp. was placed in the middle of the PDA Petri dishes. Plates were incubated in the growth chamber at 25 ± 5 °C and at 70% ± 5% relative humidity. Mycelium growth rate (mm day−1) readings were recorded after 3, 6, and 9 days. An average of two perpendicular measurements was recorded [35]. A completely randomized design (CRD) with three replicates per NaCl concentration was used.

2.4. Experiment 2: In Vitro Evaluation of Trichoderma spp. on Turfgrass Seeds under Different Levels of Salinity

Turfgrass seeds were cultured on instant agar agar medium (MERK KGaA, Darmstadt, Germany) (20 g L−1). The electrical conductivity (EC) of the medium was adjusted to three NaCl concentrations (50, 100, and 150 mM) and control (sterile-distilled water) by using sodium chloride extra pure (Scharlau), Sentmenat, Spain. Media was sterilized by autoclaving at 121 °C at 15 psi and poured in a 9 cm diameter Petri dishes under aseptic conditions.
Seeds were soaked in sterile-distilled water for 15 min, sterilized in 2.5% of commercial sodium hypochlorite solution (6%) for five minutes then rinsed three times in sterile-distilled water under aseptic conditions [36].
The spore suspension was prepared by mixing two plugs (5 mm) of 10-day-old isolates culture for each Trichoderma spp. (ThLem2017-01, TaDP2019-01, and TaDP2019-02) in 5 mL distilled water for 30 min. Spore count was adjusted to 8 × 106 spore mL−1. Seeds of turfgrass were soaked for 15 min in the spore suspension, dried in aseptic conditions [35], and plated on agar agar-medium amended with different NaCl concentrations. Ten seeds were immersed gently in each Petri dish and were incubated in the growth chamber at 25 ± 1 °C with 70% ± 5 relative humidity for 20 days. Four replicates were used for each NaCl concentration. Measurements including radicle length and coleoptile length, and the number of leaves was recorded. The Trichoderma isolate that showed best results in this assay was selected for the following experiment. Completely randomized design (CRD) with four replicates per NaCl concentration was considered. Therefore, there were 64 experimental units (10 seeds of turfgrass per each experimental unit). The readings were taken for all the seeds in each Petri dish so that 10 readings from each measurement were taken per experiment. The 10 values from each replicate were averaged to one value before statistical analysis.

2.5. Experiment 3: In-Vivo Effect of Trichoderma Isolate on Perennial Ryegrass under Different Levels of Salinity

Greenhouse experiments were conducted at the greenhouse facility, School of Agriculture, University of Jordan, Amman, Jordan (32°00′40.4″ N 35°52′20.7″ E from 27 October 2020 to 3 May 2021. The minimum and maximum average temperatures in the greenhouse were 10 °C and 25 °C, respectively.
Inoculum of Trichoderma isolate that had been selected from the second experiment was prepared on wheat as a carrier seed. Wheat seeds were rinsed and soaked in tap water for 12–24 h, loaded into sterilized mason jars and sterilized by autoclaving at 121 °C and 15 Psi. Two plugs (5 mm) of 10-day-old cultures of the selected Trichoderma isolate were added to the jars in aseptic conditions and incubated in the dark for 21 days. Jars were shaken at least once every day [37].

2.5.1. Perennial Ryegrass Cultivation and Treatments

Perennial ryegrass seeds were sown on 27 October 2020 by sowing the entire surface of the pot with approximately 100 seeds. Sterilized pots (1.4 L) were filled with sterilized, washed silica sand and were covered with 0.5 cm of wet peat moss. The growing pots are made of plastic material and each pot has five drainage holes. The soil surface area for each pot is 136 cm2 and the base area is 96 cm2. The top diameter of the pot is 13.17 cm and the base diameter is 11.06 cm. The height of each pot is 12.19 cm. A geotextile (white fabric) layer was placed in the bottom of each pot to prevent sand from passing through the pot’s holes. Pots were irrigated once every two days by pouring 500 mL of water using a beaker (500 mL) for each pot. After two weeks, pots were fertigated once a week for one month using water-soluble fertilizer 20N-20P2O5-20K2O plus trace elements. The final composition of the fertigation solution was 28.6 mmol L−1 nitrogen, 5.6 mmol L−1 phosphorus, 8.5 mmol L−1 potassium, 0.6 mmol L−1 magnesium, 0.05 mmol L−1 iron, 0.02 mmol L−1 boron, 0.04 mmol L−1 manganese, 0.03 mmol L−1 zinc, and 0.01 mmol L−1 copper pouring 500 mL of fertigated water using a beaker (500 mL) per pot. After 20 days, seven seeds of the selected Trichoderma sp. inoculum were added to each pot to inoculate the well-established perennial ryegrass plants.
After 20 days of treating pots with Trichoderma (TaDP2019-02) isolate (17 November 2020), four salinity treatments (levels) were considered (4 dS m−1 (2.9 g L−1), 8 dS m−1 (5.8 g L−1), 12 dS m−1 (8.8 g L−1), and tap water (0.30–0.90 dS m−1)) by pouring 500 mL of saline water using a beaker (500 mL) into each pot every two days.. In this experiment, pots were arranged in a completely randomized design (four levels of NaCl concentrations and two levels of Trichoderma) with five replicates.

2.5.2. Measurements

Leaves of turfgrass were mowed to a length of 7.0 cm. Clipped leaves from each pot were collected and weighed to derive the fresh weight (FW) then were oven-dried at 80 °C for 24 hr to record dry weight (DW). Dry weight was performed on a monthly basis to estimate crop production. Leaf color co-ordinates (L*, a*, and b*) were measured every two weeks by Chroma Meter measuring head (CHROMA METER CR-400, KONICA MINOLTA, Osaka, Japan). Electrical conductivity and pH of the leachate water were measured to estimate the water quality and how salt builds in soil over time. EC was measured using a pH/Conductivity Meter (Extech EC500 Waterproof EcStik П, Extech, Boston, MA, USA) and pH was measured using a pH meter (pp-203 pH meter, GOnDO Electronic Co, Taipei, Taiwan). EC and pH were recorded five times during the experiment. The pH meter was calibrated frequently between measurements by inserting the electrode in the standardizing solution buffers (first with pH 7, then with pH 4, and finally with pH 10) according to the manual protocol.
Soil respiration was determined at the end of experiment using the Solvita® kit (Solvita, CO2-Burst, Mount Vernon, ME, USA). Soil respiration is considered as an indicator of microbial activity. This test was made on two levels of salinity treatments (12 dS m−1 and control) with three replicates.
The presence or absence of the selected isolate was confirmed after six months of seeding by plating perennial ryegrass leaves and roots on PDA media. From each treatment of perennial ryegrass, five segments (5 mm) from roots and leaves were surface-sterilized in 1% hypochlorite for 3 min then rinsed five times by distilled water and plated on PDA supplemented with ampicillin. The plates were incubated at 28 °C for one week and the presence or absence of Trichoderma was determined microscopically and based on culture morphology [38].

2.6. Data Analysis

Data analyses were conducted using SAS software (Version 9.4 for Windows; SAS Institute, Cary, NC, USA). Two-way analysis of variance using general linear model PROC GLM were used for all experiments. The least significant difference test (LSD) (p ≤ 0.05) was used to identify differences between treatments [39].

3. Results

3.1. Mycelial Growth Rate of the Three Trichoderma spp. Isolates under Different Salt Concentrations

Salt stress significantly reduced mycelium growth rate of Trichoderma spp. (Figure 1). Salinity stress reduced mycelium growth rate of Trichoderma spp. by 13.8% at 200 mM, 32.4% at 500 mM, 67.7% at 750 mM and 82.4% at 1000 mM. ThLem2017-01 and TaDP2019-01 had a higher mycelium growth rate than TaDP2019-02 when salinity levels were 200, 300, 400, and 500 mM. Conversely, TaDP2019-02 had a higher mycelium growth rate at 1000 mM. Although TaDP2019-02 isolate had a lower mycelium growth rate at low levels of salinity, it remained stable at high levels of salinity comparing to ThLem2017-01 and TaDP2019-01.

3.2. In Vitro Evaluation of Trichoderma spp. on Turfgrass Seeds under Different Levels of Salinity

The growth of turfgrass seeds was significantly affected by salt levels and Trichoderma isolates. Increasing salinity levels led to a reduction of radicle length, coleoptile length, and leaf length (Figure 2). The radicle length of plants that inoculated with TaDP2019-02 isolate has increased by 91 to 575% at control compared to other isolates at the same salinity level. While at 4 dS m−1 the radicle length increased by 52 to 158% compared to the other isolates. Interestingly, TaDP2019-02 maintained high radicle length, coleoptile length, and leaf length for each level of salinity. In contrast, the ThLem2017-01 isolate had the lowest radicle length at 0, 50, and 100 mM. In addition, the TaDP2019-01 isolate had the lowest coleoptile length among all isolates and the lowest leaf length at 0, 50, 100, and 150 mM. As a result, the TaDP2019-02 isolate maintained the best morphological performance at all levels of salinity. Therefore, TaDP2019-02 isolate was selected to be used for the greenhouse experiment.

3.3. In-Vivo Effect of Trichoderma Isolate on Perennial Ryegrass under Different Levels of Salinity

Clipping FW and DW of perennial ryegrass were significantly affected by salt levels and Trichoderma treatment (Table 1). The interaction between the salinity and T. atroviride (TaDP2019-02) treatments was not significantly different. The increase in salinity levels (4, 8, and 12 dS m−1) decreased clipping FW and DW. Plants inoculated with Trichoderma had increased FW and DW of leaf clippings. FW and DW of perennial ryegrass that was inoculated with Trichoderma significantly increased by 16 to 114% and 24 to 76% than uninoculated ones, respectively (Table 1). The root mass of perennial ryegrass was more branched and longer when treated with T. atroviride (TaDP2019-02) at all levels of salinity compared to non-treated plants (Figure 3). Roots and shoots dry weight of perennial ryegrass were significantly affected by T. atroviride (TaDP2019-02) and salinity levels. When the salinity level increases the root and shoot dry weight decreases. Trichoderma atroviride (TaDP2019-02) isolate had enhanced root dry weight by 22% and shoot dry weight by 14.3% (Table 1).
The Solvita soil respiration test showed higher soil respiration (Soil-CO2-C, mg kg−1) levels in soil samples that had been inoculated with TaDP2019-02 isolate (Table 2). Soils that were inoculated with T. atroviride (TaDP2019-02) increased soil respiration by 75% compared to non-inoculated plants. There was no effect of salinity levels on CO2 concentration.
Increasing salinity levels decreased the leaf color co-ordinate ‘a*’, a greenness indicator (Table 3). However, Trichoderma (TaDP2019-02) isolate did not affect the color co-ordinate ‘a*’ of perennial ryegrass leaves. The salinity level at 12 dS m−1 increased the color ‘a*’ values by 9 to 16% than the control treatment (tap water). The interaction between the salinity and T. atroviride (TaDP2019-02) treatments was not statistically significant.

4. Discussion

The mycelium growth rate of all Trichoderma spp. decreased by increasing NaCl concentrations. The maximum colony growth was observed at 0 mM NaCl. Current results agreed with Poosapati et al. [35] who found that 50% of the isolates lost their viability when the salinity concentration reached 0.75 M NaCl. In addition, germination rates of most isolates were decreased to 60–70% at 1 M NaCl concentration. Mohamed and Haggag [40] proved that salinity stress had significant negative effects on colony growth and sporulation of wild type of T. harzianum. The response of ThLem2017-01, TaDP2019-01, and TaDP2019-02 isolates to an increase in salinity level were significantly varied among isolates. The response of mycelium growth rate to increase in salinity levels of TaDP2019-02 seems more constant than of ThLem2017-01 and TaDP2019-01 isolates. Rawat et al. [41] had tested 45 T. harzianum isolates, only five isolates tolerated salinity and grew at 240 mM NaCl. De Donno et al. [42] discovered that Trichoderma has in fact better colonized Lake Alimini Grande in Italy. These are saprophytic fungi that belong to ubiquitous species and are capable of colonising aquatic environments and brackish water. In particular, the tested Trichoderma isolates in this current experiment maintain high mycelium growth rates up to 200 mM (18 dS m−1). As a result, these Trichoderma isolates should be effective when irrigated with brackish water. Sedat et al. [43] revealed that several T. harzianum isolates were able to grow their mycelium and sporulate in the growth medium (PDA) at a concentration up to 350 mM salt. In another in vitro study, high levels of salinity (250 mM) caused morphological and physiological changes for the Trichoderma isolates [44]. According to their screening, T. harzianum (T103) was classified as the most salt-tolerant isolate [44]. In case of water reduction in the growing medium, a buildup of specific intracellular substances caused by an increase in synthetic metabolic pathways is one of the physiological changes in this kind of micro-organism [45]. The accumulated substances are neutral osmolites that can play a role as osmoprotective agents [46].
In the in vitro evaluation of the effect of Trichoderma spp. on turfgrass seeds under different levels of salinity, radicle, coleoptile, and leaf length were reduced when salinity levels increased. Salinity is an important abiotic stress that leads to negative effects on many biochemical and physiological mechanisms in seed germination, plant growth and development. In addition, salinity stress causes toxicity (ion accumulation), cell dehydration, and limit water absorption [47]. Shiade and Boelt [48] conducted an experiment on eight varieties of tall fescue at 6 to 15 dS m−1 EC. They stated that seed growth parameters (roots length, seedling, and shoots vigor index) showed a gradual decrease with increasing NaCl concentration. When salinity levels increased over 8 dS m−1, root and shoot growth of perennial ryegrass suffered seriously compared to lower salinity levels (0, 2, and 4 dS m−1) [49].
Although the mycelium growth rate of Trichoderma TaDP2019-02 isolate did not give the superior results all the time under different levels of salinity in the first laboratory experiment, plants that were inoculated with T. atroviride (TaDP2019-02) isolate maintained high radicle length, coleoptile length, and leaf length for each level of salinity. In the second experiment. T. atroviride TaDP2019-02 isolate increased seedling radicle and coleoptile length compared to a seedling that was not treated with Trichoderma sp. Trichoderma atroviride increases the percentage of germination, growth, phenolic content, and the essential oil content of salinity-stressed seedlings of fennel [50]. Lo and Lin [51] reported that cucumber plants that were treated separately with T. harzianum and T. virens strains showed a significant increase in plant height, leaf area, and root dry weight compared to non-treated seedlings. Moreover, Hoyos-Carvajal et al. [52] found that early stages of growth in bean plants were stimulated by Trichoderma strains. Trichoderma spp. promotes plant growth by enhancing nutrient uptake through increased root growth and increasing the availability of essential nutrients around the root zone [53].
In the current research, FW and DW of perennial ryegrass biomass that was inoculated with Trichoderma increased by 16 to 114% and 24 to 76% compared to uninoculated ones, respectively (Table 1). However, exposure to high levels of salinity reduced plant growth in both plants that were inoculated and non-inoculated with Trichoderma compared to plants that had non-saline treatments. The current results are consistent with previous studies which have proven that perennial ryegrass reduced growth rate and turf quality under salt stress. Decreasing in leaf growth in perennial ryegrass is considered as the earliest response to salinity stress [9,54].
The mutual symbiosis of plants with endophytic fungi can improve salt tolerance and reduce crop yield losses caused by saline conditions [55,56]. In Indian mustard, high levels of NaCl (200 mM) reduced mustard plant height by 33.7%, and plant dry weight by 34.5% [57]. However, under the same salinity conditions, shoot and plant DW for plants that were inoculated with T. harzianum increased by 13.8 and 16.7%, respectively [57]. Contreras-Cornejo et al. [58] suggested that T. virens and T. atroviride produce higher levels of indole-3-acetic acid (IAA) once it is grown in a medium supplied with 100 mM NaCl to enhance seed germination and plant growth under salt stress. Sekmen Cetinel et al. [59] found that Trichoderma citrinoviride reduced H2O2 content, which might be responsible for the protection of membrane lipids from peroxidation.
Soil respiration has been used as an indicator for soil health by measuring the CO2 emission that resulted from the respiration of soil micro-organisms [60]. Respiration gives a reliable and scientific basis on which to evaluate microbial activity [61]. In this study, the Solvita soil respiration-CO2 test proved that soils that were inoculated with Trichoderma (TaDP2019-02) had higher respiration in perennial ryegrass rhizospheres. This result is consistent with Wiedow et al. [62] who observed a significant increase in soil respiration in the treatments that were inoculated with T. saturnisporum. In addition, Yadav et al. [63] found that T. viride inoculation increased soil basal respiration.
Leaf color is one of the most crucial parameters to determine the color quality of tissue surfaces. Trichoderma (TaDP2019-02) isolate did not affect the greenness lightness ‘a’ that indicates chromaticity co-ordinates from green (−) to red (+) of perennial ryegrass leaves. High salinity levels have been reported to affect perennial ryegrass color and visual quality [64]. In addition, salt stress reduces the photosynthetic pigments due to a reduction in the synthesis of many enzymes, such as photochlorophyllide reductase and δ-aminolevulinic acid dehydratase [65]. These enzymes have a role in the impairment of ion supply (Mg2+, Fe+, and Mn2+) that are essential for the synthesis of chlorophyll [66]. In contrast, some other researchers found that high levels of salinity increased chloroplast concentration per unit in leaf area. According to their explanation, leaves became thicker and smaller [67]. Hu et al. [9] stated that chlorophyll content in perennial ryegrass had higher values with abiotic stress under greenhouse conditions. Heidari et al. [68] reported that salinity decreased chlorophyll content only in the long-term while chlorophyll content increased by increasing leaf thickness in the short-term of salinity. Inoculation of Trichoderma by seed biopriming improved wheat plants’ net photosynthesis, chlorophyll fluorescence, greenness, and stomatal conductance [69]. Khomari et al. [70] suggested that pretreatment of soybean seedlings with salt-tolerant Trichoderma repressed the decrease in leaf greenness and chlorophyll fluorescence.
This research focused on using Trichoderma isolates on turfgrass (perennial ryegrass) which is commonly used in public and residential landscapes and in sports fields. However, the robust performance of Trichoderma isolate could be applied to other related grass crops and some cereal crops and forage crops in areas that suffer from salinity stress. This should increase the forage production for the purpose of grazing, silage, and hay.

5. Conclusions

Trichoderma atroviride (TaDP2019-02) inoculation holds promise as a beneficial micro-organism and an environmentally friendly alternative to mitigate salinity. The results showed that increasing salinity levels decreased the mycelium growth rate of all Trichoderma isolates. ThLem2017-01 and TaDP2019-01 had a higher mycelium growth rate than TaDP2019-02 when salinity levels were 200, 300, 400, 500 mM while TaDP2019-02 had a higher mycelium growth rate at 1000 mM. The response of mycelium growth rate to an increase in salinity levels of TaDP2019-02 was more constant than the response of ThLem2017-01 and TaDP2019-01. In the second experiment, the results revealed that Trichoderma inoculated-seedlings had higher radicle length, coleoptile length, and leaf length than uninoculated plants. Mainly, TaDP2019-02 isolate maintained the best morphological performance at all levels of salinity. Therefore, TaDP2019-02 isolate was selected to be used for further assessment in the greenhouse experiment. In this experiment, perennial ryegrass clippings FW and DW that were inoculated with TaDP2019-02 isolate were 16 to 114% and 24 to 76% higher than uninoculated ones, respectively. In addition, soils that were inoculated with Trichoderma (TaDP2019-02) had higher CO2 respiration (75%) than the control. T. atroviride (TaDP2019-02) inoculation holds promise as a beneficial micro-organism and an environmentally friendly alternative to mitigate salinity. Therefore, applying Trichoderma isolate to turfgrass and other related grasses has the potential to enhance turfgrass quality and even increase forage production in regions with saline stress.

Author Contributions

Conceptualization, M.G.A.-A. and K.M.A.; methodology, N.S.A.-S.; validation, M.G.A.-A., K.M.A. and Y.A.O.; formal analysis, M.G.A.-A.; investigation, N.S.A.-S.; resources, N.S.A.-S. and M.G.A.-A.; data curation, N.S.A.-S.; writing—original draft preparation, N.S.A.-S.; writing—review and editing M.G.A.-A., K.M.A. and Y.A.O.; supervision, M.G.A.-A. and K.M.A.; project administration, M.G.A.-A.; funding acquisition, M.G.A.-A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was fully funded by the Deanship of Scientific Research at the University of Jordan, Funding number 19/2020/961.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Acknowledgments

The authors would like to thank the Deanship of Scientific Research at the University of Jordan, Jordan, for the full funding of this project.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Water 14 03943 g001 550
Figure 1. The mycelium growth rate of the three isolates of Trichoderma spp. under different NaCl concentrations (50, 100, 150, 200, 300, 400, 500, 750, and 1000 mM) (they are equivalent to 4, 8, 12, 18, 28, 38, 44, 64, and 84 dS m−1). Sterile-distilled water was used as a control. Note that 1000 mM = 58.44 g of NaCl per one litre. Measurements were taken after three, six and nine days of subculturing on PDA media. Closed and open symbols represent the mean ± SE for different Trichoderma isolates (n = 3). Error bars represent standard error of the mean.
Figure 1. The mycelium growth rate of the three isolates of Trichoderma spp. under different NaCl concentrations (50, 100, 150, 200, 300, 400, 500, 750, and 1000 mM) (they are equivalent to 4, 8, 12, 18, 28, 38, 44, 64, and 84 dS m−1). Sterile-distilled water was used as a control. Note that 1000 mM = 58.44 g of NaCl per one litre. Measurements were taken after three, six and nine days of subculturing on PDA media. Closed and open symbols represent the mean ± SE for different Trichoderma isolates (n = 3). Error bars represent standard error of the mean.
Water 14 03943 g001
Water 14 03943 g002 550
Figure 2. Radicle length (A), coleoptile length (B), and leaf length (C) readings after 20 days under different NaCl concentrations (50, 100, and 150 mM) (they are equivalent to 4, 8, and 12, dS m−1). Sterile-distilled water was used as a control. Note that 1000 mM = 58.44 g of NaCl per one litre. Lowercase letters that share different letters are significantly different according to the least significant difference (LSD) test (p ≤ 0.05). Closed and open symbols represent the mean ± SE for different Trichoderma spp. isolates (n = 4). Error bars represent standard error of the mean.
Figure 2. Radicle length (A), coleoptile length (B), and leaf length (C) readings after 20 days under different NaCl concentrations (50, 100, and 150 mM) (they are equivalent to 4, 8, and 12, dS m−1). Sterile-distilled water was used as a control. Note that 1000 mM = 58.44 g of NaCl per one litre. Lowercase letters that share different letters are significantly different according to the least significant difference (LSD) test (p ≤ 0.05). Closed and open symbols represent the mean ± SE for different Trichoderma spp. isolates (n = 4). Error bars represent standard error of the mean.
Water 14 03943 g002
Water 14 03943 g003 550
Figure 3. The root mass of perennial ryegrass with and without T. atroviride (TaDP2019-02) under different salinity levels (0, 4, 8, and 12 dS m−1). The white areas between roots are a white fabric filter (geotextile) to prevent sand from filtering out of the pots.
Figure 3. The root mass of perennial ryegrass with and without T. atroviride (TaDP2019-02) under different salinity levels (0, 4, 8, and 12 dS m−1). The white areas between roots are a white fabric filter (geotextile) to prevent sand from filtering out of the pots.
Water 14 03943 g003
Table
Table 1. ANOVA of clipping fresh and dry weight and shoot and root dry weight for perennial ryegrass with and without (control) T. atroviride under different levels of salinity (0, 4, 8, and 12 dS m−1).
Table 1. ANOVA of clipping fresh and dry weight and shoot and root dry weight for perennial ryegrass with and without (control) T. atroviride under different levels of salinity (0, 4, 8, and 12 dS m−1).
Main Effect Clipping Dry Weight (g)Shoot Dry Weight (g)Root Dry Weight (g)
Day 8811918588119185190190
Trichoderma (T)
With 1.51 a 12.21 a12.10 a0.35 a0.46 a3.37 a19.64 a8.22 a
Without 2 1.12 a1.35 b10.31 b0.26 a0.26 b2.71 b17.20 b6.77 b
LSD 0.42630.36291.54320.09780.08820.40922.02741.0278
Salinity (S)
0 2.34 a2.81 a17.84 a0.52 a0.57 a4.64 a22.56 a13.29 a
4 1.34 b1.89 b11.62 b0.34 b0.40 b3.16 b18.06 b8.60 b
8 0.84 bc1.33 c10.05 b0.20 bc0.27 bc2.79 b17.79 b4.76 c
12 0.72 c1.07 c5.30 c0.17 c0.21 c1.57 c15.99 b3.79 c
LSD 0.60290.51322.18240.13830.12480.57872.83781.4477
ANOVA
T 0.0716<0.00010.02460.0701<0.00010.00260.02050.0055
S <0.0001<0.0001<0.0001<0.0001<0.0001<0.00010.0009<0.0001
T × S 0.78290.36430.77240.69760.39130.25260.81920.7262
Note(s): 1 Numbers followed by different letters within a column are significantly different according to the least significant difference (LSD) test (p ≤ 0.05). 2 Treatment that did not receive Trichoderma inoculation (without) and had a zero salinity level was considered as a control treatment.
Table
Table 2. ANOVA of CO2 levels for perennial ryegrass soil samples with and without T. atroviride (TaDP2019-02) under two levels of salinity (0 and 12 dS m−1).
Table 2. ANOVA of CO2 levels for perennial ryegrass soil samples with and without T. atroviride (TaDP2019-02) under two levels of salinity (0 and 12 dS m−1).
Main EffectCO2%
Trichoderma (T)
With2.67 a 1
Without 2 1.50 b
LSD0.3842
Salinity (S)
02.25 a
122.92 a
LSD0.3843
ANOVA
T0.0066
S0.2598
T × S0.4876
Note(s): 1 Numbers followed by different letters within a column are significantly different according to least significant difference (LSD) test (p ≤ 0.05). 2 Treatment that did not receive Trichoderma inoculation (without) and had a zero salinity level was considered as a control treatment.
Table
Table 3. ANOVA of color parameters for perennial ryegrass with and without T. atroviride (TaDP2019-02) under different levels of salinity (0, 4, 8, and 12 dS m−1).
Table 3. ANOVA of color parameters for perennial ryegrass with and without T. atroviride (TaDP2019-02) under different levels of salinity (0, 4, 8, and 12 dS m−1).
Main Effect L* 1a* 2b* 3
Day284358284358284358
Trichoderma (T)
With 34.72 a 434.39 a34.69 a−15.65 a−14.27 a−13.68 a23.42 a22.40 a22.19 a
Without 5 36.13 a34.88 a35.65 a−15.07 a−13.90 a−13.00 a23.23 a21.54 a21.67 a
LSD 1.60011.33121.13350.69140.88860.79461.19550.87310.7747
Salinity (S)
0 37.69 a36.23 a36.15 a−16.50 b−15.07 b−14.66 c24.26 a23.20 a22.81 a
4 36.38 ab35.15 a35.52 ab−15.36 a−14.22 ab−13.26 b23.61 ab21.79 bc22.39 a
8 34.58 bc34.73 a34.79 ab−14.89 a−13.76 a−13.59 bc22.44 ab22.16 ab21.98 a
12 33.04 c32.43 b34.23 b−14.70 a−13.39 a−11.86 a22.99 b20.75 c20.52 b
LSD 2.26291.88261.60310.97781.25671.12381.69081.23481.0956
ANOVA
T 0.08110.45520.09320.09660.39950.09140.75210.05270.1823
S 0.00120.00240.09990.00320.04460.00020.16990.00330.0011
T × S 0.32450.03330.25160.84230.91240.07560.64260.01030.6053
Note(s): 1 ‘L*’ indicates lightness. 2 ‘a*’ indicates chromaticity co-ordinates from green (−) to red (+). 3 ‘b*’ indicates chromaticity co-ordinates from blue (−) to yellow (+). 4 Numbers followed by different letters within a column are significantly different according to the least significant difference (LSD) test (p ≤ 0.05). 5 Treatment that did not receive Trichoderma inoculation (without) and had a zero salinity level was considered as a control treatment.
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Abu-Shanab, N.S.; Alananbeh, K.M.; Othman, Y.A.; Al-Ajlouni, M.G. Effect of Using Trichoderma spp. on Turfgrass Quality under Different Levels of Salinity. Water 2022, 14, 3943. https://doi.org/10.3390/w14233943

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Abu-Shanab NS, Alananbeh KM, Othman YA, Al-Ajlouni MG. Effect of Using Trichoderma spp. on Turfgrass Quality under Different Levels of Salinity. Water. 2022; 14(23):3943. https://doi.org/10.3390/w14233943

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Abu-Shanab, Nour S., Kholoud M. Alananbeh, Yahia A. Othman, and Malik G. Al-Ajlouni. 2022. "Effect of Using Trichoderma spp. on Turfgrass Quality under Different Levels of Salinity" Water 14, no. 23: 3943. https://doi.org/10.3390/w14233943

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