# Identification of beneficial Lebanese Trichoderma spp. wheat endophytes

Naeif Matar, Catherine Macadre, Gamal a G Ammar, Alexis Peres, Boris Collet, Naim El Boustany, Loïc Rajjou, Falah As-Sadi, Marie Dufresne, Pascal Ratet

► To cite this version:

Naeif Matar, Catherine Macadre, Gamal a G Ammar, Alexis Peres, Boris Collet, et al. Identification of beneficial Lebanese Trichoderma spp. wheat endophytes. Frontiers in Plant Science, In press, 13, 10.3389/fpls.2022.1017890. [hal-03876005](https://hal-03876005)

HAL Id: hal-03876005

https://cnrs.hal.science/hal-03876005v1

Submitted on 28 Nov 2022

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# Identification of beneficial Lebanese *Trichoderma* spp. wheat endophytes

1 Naeif Matar¹,², Catherine Macadre¹, Gamal A. G. Ammar¹,³, Alexis Peres¹, Boris Collet⁴, Naim

2 El Boustany⁵, Loïc Rajjou⁴, Falah As-Sadi²,⁶, Marie Dufresne¹, Pascal Ratet¹

3 ¹ Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2),
4 91405, Orsay, France.

5 ¹ Université de Paris, Institute of Plant Sciences Paris-Saclay (IPS2), F-91405 Orsay, France

6 ²Department of Life & Earth Sciences, Faculty of Sciences I, Laboratory of Microbiology, Lebanese
7 University, Beirut, Lebanon.

8 ³ Biotechnology Unit, Plant Production Department, Arid Lands Cultivation Research Institute, City
9 of Scientific Research and Technological Applications, Alexandria 21934, Egypt.

10 ⁴ Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB), Versailles,
11 France.

12 ⁵Department of Biology, Faculty of Sciences I, Lebanese University, Beirut, Lebanon.

13 ⁶ The Lebanese University, Faculty of Agronomy, Dekwaneh, Beirut, Lebanon.

15 * Correspondence:
16 pascal.ratet@universite-paris-saclay.fr

18 Abstract

19 Wheat is one of the most important crops in the world. Its production can be influenced by a diversity
20 of beneficial and pathogenic rhizospheric microbes, including fungi. Amongst them, beneficial
21 *Trichoderma* spp. can be used as alternatives to chemical fertilizers, as they are cheaper and harmless
22 to the environment. Our study aimed to isolate, identify, and characterize *Trichoderma* spp. from
23 Lebanon associated to wheat. Two *Trichoderma* strains belonging to *T. afroharzianum*, and *T.
24 guizhouense* species, were isolated and found to be endophytes enhancing root growth and producing
25

IAA. Inoculation also improved seedling development, and boosted general production. These
*Trichoderma* spp. have thus the capacity to be used as organic fertilizers for wheat.

26

27

28

29

**Keywords: Trichoderma spp., Endophyte, Auxins, 3-indole-acetic acid, Plant root growth promotion, Wheat yield.**

30

31

**Introduction**

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

Wheat (*Triticum* sp. L.) is one of the most important crops in the world, ranking third behind corn and rice, while providing the basic nutrients (carbohydrates, proteins, vitamins, minerals, and fibers) to humans (Asseng et al., 2011). The world population is projected to exceed 8 billion by the end of 2022, and the demand for wheat is expected to exceed 880 million metric tons by 2050 (https://www.fao.org/worldfoodsituation/csdb/en/). Wheat culture has thus to be widened for overcoming world increasing demand. In addition, recent challenging crisis like Covid-19 (coronavirus disease 2019) pandemic and the world conflicts negatively impact wheat production and will probably threaten wheat markets in the near future (Glauben et al. 2022; ASCAME, 2022). On the other hand, communities are requesting for sustainable, cleaner, and harmless production approaches to avoid more climate change development. In this context, alternative strategies have to be followed, and finding new safer solutions is necessary in order for example to reduce the widespread use of chemical fertilizers which negatively affect earth's natural resources. As a result, recent studies aimed at improving biofertilizers and plant bio stimulants using beneficial bacteria, yeasts, or fungi. Among fungi, *Trichoderma* spp. are ubiquitous free-living fungi (Almi & Dehimat, 2016; Esposito & da Silva, 1998) of the *Hypocreaceae* family, occurring widely in all soils (Ghazanfar et al., 2018) and representing major components of the soil mycoflora (Kubicek et al., 2003; Widden & Abitbol, 1980). These ascomycete fungi, which inhabit root ecosystems, grow and proliferate using carbohydrates secreted by plant roots. They also behave as endophytes, penetrating the epidermis of the root tissue (rhizodermis), and few cell layers below (Topolovec-Pintarić, 2019). They proliferate better at mesophilic temperatures (15-35 °C) (Carro-Huerga et al., 2021) and tolerate a wide pH range (Topolovec-Pintarić, 2019). These fungi improve plant photosynthetic capacity, and induce defenses (Vargas et al., 2009, 2011) and many stress-sensitive genes (Alfano et al., 2007; Brotman et al., 2012; Morán-Diez et al., 2012). *Trichoderma* are among the best studied biological control microbes, and are marketed as active ingredients in biofertilizers (plant growth promoters), biopesticides, bioremediates,
and natural resistance stimulants. Consequently, their application reduces production costs, and
negative environmental impact (Dennis and Webster 1971a; Dennis and Webster 1971b; Harman and
Kubicek 2002; Ghazanfar et al. 2018). *Trichoderma* fungi improve plant growth through the
solubilization of nutrients, and therefore have better effects under nutritional and abiotic stress
conditions (Mastouri et al., 2010, 2012; Shoresh et al., 2010). The use of *Trichoderma* in seed coating
also improves physiological stresses such as aging, and seed dormancy (Delgado-Sánchez et al. 2010;
Mastouri et al., 2010). The plant hormone auxin controls many aspects of development including
plant tissue growth, cell division, cell differentiation, and protein synthesis. The plant naturally
produced auxin IAA that can be also produced by microorganisms like fungi including Trichoderma.

In this study we identified and characterize two *Trichoderma* strains from Lebanon and tested their
ability to promote wheat growth.

67

## 68 **Materials and methods**

69 **Plant material:**

The wheat cultivar ‘Apogee’ was used for fungal characterization (G. Li *et al.* 2017; Gatti *et al.* 2018).
Apogee has a relatively brief life cycle under long days, and without vernalization, it flowers 25 days
after planting.

73 **Soil and plant sampling:**

Samples in this study were collected on May 2020 (late spring and early summer) from three different
sites in Lebanon namely: 1) The international center for agricultural research in the dry areas
(ICARDA) -Terbol; 33.81263953421374, 35.99166470541601, 2) Lebanese agricultural research
institute (LARI) – Tal Amara; 33.86500666714277, 35.984715713142634, and 3) Doueir;
33.39043029381532, 35.417987723093724. For every location, five samples from soil, together with
five plant samples were screened for *Trichoderma spp.* .

Soil samples were taken from the soil section called "horizon", using a 0 to 15 cm top layer shovel,
representing the surface area of the profile, where nutrients and most of the roots are present, and where
nutrient absorption takes place. This also corresponds to the area where *Trichoderma* spp. are generally
found. Sampling was carried out within a radius of 10 to 30 cm from the base of the plant depending
on the case. Each sample was transferred in an appropriate plastic bag with its reference (place, date,
85

86

87

sample number, etc.). Five samples were collected, and homogenized before the final packaging of approximately 1 kg of the sample, placed in plastic bags (to avoid contamination) was kept frozen (-20°C).

88

89

For plant sampling, 5-6 independent plant samples (root and stem) spaced at least 10 m apart were placed in plastic bags and kept frozen (-20°C).

90

## Fungus isolation from soil:

91

92

93

94

95

1 g of soil was homogenized in 10 ml sterile distilled water by thorough vortexing, then filtrated onto sterile Miracloth®. The filtrate was then plated onto 9 cm diameter petri dishes (100 ul filtrate plated per dish), containing potato dextrose agar (PDA) amended with rose Bengal (0.15 g/L), pentachloronitro-benzene (0.15 g/L), chloramphenicol (34 µg/ml), and ampicillin (100 µg/ml). One petri dish was plated per soil mixture.

96

## Fungus isolation from plants:

97

98

99

100

101

102

103

104

105

106

107

108

The internal colonization of roots or stems by potential *Trichoderma* sp. was detected following surface sterilization of approximately 2 cm root fragments for 10 sec in 95% ethanol, rinse in sterile water for 10 sec, submersion for 20 sec in 2.5% sodium hypochlorite, then washed three times (2 times for 20 sec, and a third time for 60 sec) in sterile water. Fragments were then let dry under laminar flow on sterile filter. 0.5 cm fragments were placed on PDA amended with rose Bengal, pentachloronitro-benzene, chloramphenicol and ampicillin. Plates were incubated at 24°C in the dark for 4-6 days, until fungal development. After 4 days a large number of fungal strains appeared and were transplanted (1st transplanting incubate at 26°C). Later, 10 days old, pre-incubated Petri dish (26°C) was selected, in order to make the 2nd transplant on PDA complemented with Rose Bengal (for Soil and Plant samples). A week after, purification was done by scraping half a colony, depending on the growth rate. Purification was done by plating 1/10, 1/100 or 1/1000 dilutions on PDA complemented with Rose Bengal. After 7 days, the fungal strains were transplanted on PDA and incubated at 26°C.

109

## DNA isolation, sequencing and phylogenetic analyses:

110

111

112

113

114

**DNA isolation:** Liquid culture were done in potato dextrose broth (PDB) medium (50ml), with 5 plugs (agar + fungi) for 3 days (under agitation) according to (Atoui et al., 2012), with modifications as follows: for approximately 200 mg of powder (ball or mortar grinding). 1 mL of extraction buffer (200 mM Tris - HCl, pH 7.5; 288 mM NaCl; 25 mM EDTA, pH8.0; 0.5% SDS) was added and vortexed vigourously till homogenization. Tubes were centrifuged at 12,000 rpm for 5 minutes (4°C). 750 µL
were then transferred to a new tube, and 215 µL of 3M potassium acetate were added, mixed and incubated on ice for 30 min, centrifuged at 12,000 rpm for 15 min at 4°C. 700 µL of the supernatant were collected, transferred to a new tube and 500 µL of cold isopropanol (-20°C) were added. Tubes were centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was removed, and the pellet washed with 500 µL of cold 70% ethanol (-20°C) for 5 min (4°C). The tubes were vortexed, and centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatant was removed, the pellet was briefly dried, and resuspended in 200 µL of sterile milliQ H2O + 1 mg/mL RNase A (1/10 stock solution at 10 mg/mL). The sample was incubated for 1h at 37 °C, and DNA concentration was done using NanoDrop. As control, 2 to 5 µL of each sample was run on 0.8% agarose gel.

**DNA sequencing:** Further *Trichoderma* identification was done by partial sequencing of the transcribed spacer sites (ITS) (550 bp), the Calmoduline gene (CAL) (458 bp) and the Elongation Factor (EF) gene (350 bp). The oligonucleotides used for amplification and sequencing are: for the calmoduline (CAL-228F: GAGTTCAAGGAGGCCTTCTCCC / CAL-737R: CATCTTTCTGGCCATCATGG), for the elongation factor gene (EF1-728F: CATCGAGAAGTTCGAGAAGG/ EF1-986R: TACTTGAAGGAACCCTTACC), and for the internal transcribed space (ITS1: TCCGTAGGTGAACCTGCGG/ITS4: TCCTCCGCTTATTGATATTGC). The nucleotide sequences were compared with those deposited in GenBank using the BLAST program on the NCBI website.

The phylogenetic tree was constructed using the MEGA-X program (Kumar et al., 2018). The sequences used correspond to a concatemer of the CAL, EF and ITS1 sequences. Supplementary Table (1) shows detailed supplementary information for the strains used in the phylogenetic analyses, with their corresponding geographic origin, substrate, voucher number, and GenBank accession numbers. Briefly, the evolutionary history was inferred by using the Maximum Likelihood method, and Tamura-Nei model (Tamura & Nei, 1993). The bootstrap consensus tree, inferred from 1000 replicates (Felsenstein, 1985), is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions, reproduced in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees, in which the associated taxa clustered together in the bootstrap test (1000 replicates) were shown next to the branches (Felsenstein, 1985). Initial tree (s) for the heuristic search were obtained automatically by applying Neighbor-Join, and BioNJ algorithms, to a matrix of pairwise distances, and were estimated using the Tamura-Nei model, and then the topology, with superior log likelihood value was selected. This analysis involved 26 nucleotide sequences.

**Morphological characterization of the *Trichoderma* strains:**
146

The fungal strains were maintained on PDA with glucose at 26°C for 7 days. Microscopic visualization of *Trichoderma* conidiophores was done using fungi grown for 3 days on a PDA plate complemented with glucose (Glc).

147

148

149

**Endophytism tests:**

150

The endophytic capacity was tested using the Apogee spring wheat variety. The endophytism behavior of the 2 *Trichoderma* strains was examined using confocal microscopy. Two methods of inoculation were used and described below as the PDA inoculation method and the spore inoculation method. This experiment was repeated twice, and 5 samples were taken for each condition (with and without *Trichoderma*) for analysis.

151

152

153

154

155

For the PDA inoculation method, the seed sterilization protocol was modified from Jaber (2018). Seeds were soaked for 5 min with bleach (0.6% sodium hypochlorite) with stirring (150-200 rpm), then rinsed three times for 10 min with sterile milliQ water. They were then stratified for 3 to 5 days in the dark at 4°C, in distilled sterilized water. The inoculation was done on a PDA with 1% Ampicillin (100 mg/ml)/1% Chloramphenicol (34 mg/ml) dish. Five wheat seeds were spread over a circle of 3 cm diameter and cultured at 26 °C and in the dark. 24 hours later, a *Trichoderma* plug was added (7 days of culture on PDA) in the center of the petri dish and incubated at 26°C. Finally, roots were harvested after 5 days of culture.

156

157

158

159

160

161

162

163

For the spore inoculation method, the seed sterilization was carried out as mentioned above. The coating of the seeds with *Trichoderma* spores was then done as follow: the seeds and a spore solution (three 7 days old Trichoderma petri dishes were used, where germinated spores were rubbed off by distilled sterilized water, then filtered to collect a free-hyphae spore solution, and finally measure the concentration using Thoma's-cell slide to get a 10⁷ spores / 10ml final concentration. The solution was then shacked for 20-30 min, then dried for a few minutes in the hood, before sowing. Seeds were then sown in glass tubes in ½ MS (2.2g / L) with phytagel (2g / L), about 5mm deep, to prevent the roots from remaining on the surface.

164

165

166

167

168

169

170

171

*Root samples treatment for imaging:* After 5 days of incubation, roots were collected, gently cleaned from the excess of external fungal colonization, cut in 1.5 / 2.0 cm length, and washed with water or PBS 1X: 20 min. Root fragments were fixed overnight in a mixture of 95% Ethanol - 100% acetic acid (3: 1) (v: v). Samples were then stabilized 1h stabilization 1X PBS.

172

173

174
175 Roots were included in 1X PBS with 3% agarose for 5 min ("Seaken LE Agarose for electrophoresis")
176 (0.6 g of agarose in 20 mL). For staining, cross-sections were immersed in the staining solution (20
177 µg/mL propidium iodide; 10µg/mL WGA-Alexa Fluor 488, 0.02% Tween 20 made up in 1X PBS) for
178 2 min, immersed in water for few seconds to remove the staining solution excess. Cross sections of
179 wheat roots were visualized to check endophytism using Confocal microscope (Zeiss LSM880).

180

181 **In planta** wheat yield evaluation:

182 To evaluate wheat yield under controlled conditions, 10 days old plantlets, from seeds, pre-cultured on
183 both, PDA (PDA approach), and phytagel ½ MS media (tube approach), were used. 32 plantlets from
184 each treatment (with, and without *Trichoderma*) were transplanted to sterilized peatmoss soil
185 (www.jiffygroup.com- Substrates for the professional horticulture) with perlite (3:1). Wheat yield and
186 yield components, including number of spikes/plants, number of grains/plants, grain weight/plant (g),
187 whole plant dry weight (g), hundred grain weight (g), and harvesting index (HI%). were then measured.

188 All plants were kept in a growth chamber, under 16h: 8h light to dark period, with 265 µE m⁻² s⁻¹, at
189 22 °C, and 20 °C, day to night temperatures, and 60% relative humidity (RH). Regular irrigation was
190 practiced, in parallel with fertilization practices at 14:12:32 N:P:K, plus 41 g Hortrilon engrais
191 micronutrients (B 0.5%, Cu 2.5%, Fe 5.0%, Mn 2.5%, Mo 0.5%, and Zn 0.6%) (https://www.fertil.fr/).

192 Statistical analysis:

193 Statistical analysis was conducted for the treatments, on a completely randomized design (CRD), and
194 a factorial completely randomized design, with 32 replicates. The R software (R 3.3.4; R development
195 core team, 2017) was used to analyze experimental data, calculate means, standard deviations, bar
196 charts, and interaction, using the R package "ggplot2" (Wickham, 2009). Analysis of variance
197 (ANOVA) was performed using the "lmerTest" R package (Kuznetsova et al., 2017).

198 To test the effect of the *Trichoderma* strains on wheat root development, *Trichoderma* spores (10⁷
199 spores/10 ml) were incubated with wheat seeds in a tube containing 0.5X MS medium (Kapusi &
200 Stoger, 2022) with phytagel without sugar. 10 days after germination, we measured the primary root
201 growth.

202 Trichoderma auxin production:
203
204

Auxin production was measured using the Gusmiaty method (Gusmiaty et al., 2019) with some modifications in presence or absence of tryptophan.

205
206
207

Briefly, five plugs of each *Trichoderma* strain with, and without tryptophan were incubated in 0.5X MS liquid medium (2151,45 mg/L), 2-(N-morpholino) ethanesulfonic acid (MES) (0.25 g/L), glucose 2%, pH = 5.78 adjusted with 1M KOH (3 biological repeats).

208
209
210
211
212

To determine the auxin concentration (IAA in our case), Salkowski reagent, and IAA standard curve were done, according to (Gusmiaty et al., 2019). A standard IAA solution curve showing the relationship between the standard IAA solution (x), and its absorbance (y), was made, and allowed the quantification of IAA production by *Trichoderma* using the following equation: Y= a+ bX (a = Intersep, b = Slope (Regression Coefficient), Y = Absorbance, X = Concentration).

213

Preparation of Total Protein Extracts and SDS-PAGE:

214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229

Total protein extracts were prepared from wheat grains grounded in liquid nitrogen using mortar and pestle. From 50 mg of flour, total soluble proteins were extracted at room temperature in 960 µl thiourea/urea lysis buffer (Harder et al., 1999) containing 7 M urea, 2 M thiourea, 6 mM Tris-HCl, 4.2 mM Trizma® base (Sigma-Aldrich, Lyon, France), 4% (w/v) CHAPS) supplemented with 162 µl of the protease inhibitor cocktail Complete Mini (Roche Diagnostics France, Meylan, France). Then, 22.5 µl of dithiothreitol (DTT, 1 M, Sigma-Aldrich), 4 µl of DNase I (Roche Diagnostics) and 10 µl of RNase A (Sigma-Aldrich) were added to the sample. For each sample, the protein extract was stirred for 30 min at 4°C and then centrifuged (35,000g, 15 min) at 4°C. The supernatant was submitted to a second clarifying centrifugation as above. The final supernatant corresponded to the total protein extract. Protein concentration in each extract was measured according to Bradford (1976). Bovine serum albumin was used as a standard. Separation of proteins was performed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with a 4% (w/v) polyacrylamide stacking gel and a 12% (w/v) polyacrylamide resolving gel as described by Laemmli (1970). Electrophoresis was carried out at a constant current of 110 volts (10 mA) for 2 h using a Tris-glycine running buffer with of 0.2%. After electrophoresis, proteins were stained with GelCode® Blue Stain Reagent (Thermo Scientific, Rockford, IL).

230

231

Results
232 **Characterization of two Lebanese *Trichoderma* strains isolated from wheat**

233 In order to isolate *Trichoderma* fungi associated to wheat in Lebanon, 40 fungal isolates were isolated
234 in total from three sampling sites. 21 were isolated from soil and 19 were isolated from plants. DNA
235 was prepared from them and the ITS sequences amplified and sequenced. Based on the ITS sequences
236 from these 40 colonies, 2 fungal isolates of the genus *Trichoderma* were identified (Supplementary
237 Table 2). The two isolates were derived from site 3 (Doueir) and were named S3PA (isolated from
238 stem base), and S3SB (isolated from soil). The other isolates included fungi from the genus: *Alternaria*,
239 *Aspergillus*, *Boeremia*, *Cladorrhinum*, *Fusarium*, *Macrophomina*, *Microdochium*, *Mucor*, *Penicillium*,
240 *Stromatinia*, and *Talaromyces* (Supplementary Table 2).

241 A more precise molecular description of these two *Trichoderma* fungi was done by characterizing the
242 partial sequences of their Elongation factor (EF-1) and Calmodulin (Cal) gene sequences. The three
243 partial sequences (ITS-EF-1 and Cal) were then concatemerized (550, 350, and 489 bp, respectively)
244 and compared to the same concatemerized sequences of 14 *Trichoderma* species (Supplementary Table
245 1) from the public database and representing the *Trichoderma* diversity. The result of this analysis
246 (Figure 1) showed that the two isolates probably belong to the two species *T. afroharzianum* and *T.
247 guizhouense* belonging to the *Harzianum* species complex.

248 **Morphological characterization of the two Lebanese *Trichoderma* strains**

249 After 48h at 26°C on PDA, the *T. guizhouense* formed an abundant aerial mycelium, cottony and
250 radiating a white color. At 72 h, the formation of a greenish ring was observed, which increased by
251 time of incubation, and diffused in the media, until covering almost the whole petri dish at 168 h. The
252 conidia were visualized from 48h in broad concentric bands (green), in the aerial hyphae (Figure 2A).
253 After 72h of growth on PDA (26°C), *T. afroharzianum* produced an abundant aerial mycelium, cottony,
254 radiating a white color (Figure 2B). A dense hyphae growth in the petri dish center was observed after
255 transplantation of *Trichoderma*, while 96 h later, the formation of a greenish ring was observed, which
256 increased with the time of incubation, and diffused in the media, until covering almost the whole petri
257 dish at 168 h.

258 We then morphologically characterized the two newly isolated strains. After 3 days, pyramidal
259 conidiophores were repeatedly branched, irregularly arranged in whorls, and showed clusters of
260 divergent, usually unevenly folded, barrel shaped, to nearly sub-globose phialides. The ellipsoidal to
261 globose conidia tend to be greenish to light turquoise, sometimes hyaline to cluster in aggregates at the
terminal of the phialides (Zhu & Zhuang, 2015). Conidiophores of *T. guizhouense*, showed opposite branches. The main axis and each branch terminated at a cruciate whorl of 3 ampulliform phialides (Figure 2C). Conidia, appearing within 48-72 h, were typically abundant, and disposed in one, or three concentric rings, around the point of inoculation. Green pigment sometimes diffused in the medium (Figure 2B). After 3 days of incubation on PDA, the pyramidal conidiophores of *T. afroharzianum* showed opposite branches. The main axis, and each branch terminated at a cruciate whorl of 3 ampulliform phialides green to dark green when aging (Figure 2D).

The two isolated strains thus grow rapidly on PDA medium with phenotypic characteristics of other *Trichoderma* stains (Chaverri et al., 2015).

## The Lebanese *Trichoderma* strains are wheat root endophytes

*Trichoderma* fungi were previously described as endophytes (Tseng et al., 2020). In order to know if the two Lebanese strains isolated from wheat behave also as endophytes, two modes of inoculation were tested, either by inoculating the seeds by co-cultivation on PDA medium (PDA inoculation method), either by spore coating of the seeds and germination of the seeds *in vitro* (spore inoculation method, see Material and Method section). In both cases the root sections of the plants were stained with propidium iodide and WGA-Alexa Fluor 488, staining the fungus in green. These sections were observed using confocal microscopy. Using both inoculation methods, fungal hyphae were observed inside the root for the two *Trichoderma* strains (Figure 3). The infection level observed using the PDA inoculation method seemed to be higher than the spore inoculation method that had lower concentration of the spore solution. In addition, *T. guizhouense* infected rhizodermis and cortex cells using the first methods (Figure 3A, B) and mostly rhizodermis using spore coating (Figure 3 E, F). The *T. afroharzianum* strain appears less efficient in root colonization, invading mostly the rhizodermis cell layer using the PDA inoculation (Figure 3.C, D) and colonizing only the root surface using spore coating (Figure 3 G, H). Using leaves of 4 weeks old plants, we were unable to reisolate the inoculated fungi, suggesting that these *Trichoderma* strains are not able to colonize the aerial part of the plant.

Altogether this experiment shows that the two strains can behave as fungal wheat root endophytes but that *T. guizhouense* can invade more efficiently root tissue as compared to *T. afroharzianum* and that the inoculation method can influence the level of colonization.

## The two *Trichoderma* strains promote wheat root growth and wheat aerial development and produce auxin
292 *Trichoderma* strains have been often used as plant growth promoting microorganisms (Tseng et al., 2020). In order to know if the two strains studied here can also behave as plant growth promoting fungi (PGPF), we tested their growth promotion effect on young seedlings *in vitro*, using the spore coating method and ½ MS medium without sucrose.

296 This experiment shows that the presence of the two *Trichoderma* strain resulted in a significant increase of the primary, and secondary root growth, as well as the aerial part of the plant (Figure 4A).
297 Total root, and aerial length (cm) of inoculated wheat plants, with and without *T. guizhouense* and *T. afroharzianum* respectively, is shown in Figure 4B. The results indicated that plants which were incubated with either *T. guizhouense* or *T. afroharzianum* had increased root growth and aerial development as compared to non-inoculated plants. Root length was increased by 37.6% and 37.7% for plants inoculated by *T. afroharzianum* and *T. guizhouense* respectively. For the aerial part we observed a 5.2% increase in length with both fungi. In this experiment, the inoculation by the *Trichoderma* strains enhanced principal root length (55%), total root length (70%), and aerial part lengths (35%) as well as total aerial plant weight (Figure 4B and Supplementary Figure 1A).

301 302 303 304 305 Total root, and aerial mass of wheat plants, with and without *T. guizhouense* and *T. afroharzianum* respectively are shown in Supplementary Figure 1C. The inoculation with *T. afroharzianum* resulted in an increased of 61% for the total root mass and 41%, for total aerial mass, while the treatment with *T. guizhouense* resulted in an increase of 63% for total root mass and 43%, for the aerial mass.

310 We further measured the principal root length of inoculated wheat plants over a 10 days period (kinetics), with and without *T. guizhouense* or *T. afroharzianum*. The root growth promoting effect of the two strains was already detectable 2 days after sawing and continued to increase over the 10 days period (Supplementary Figure 1A).

315 The increased root growth observed in presence of the two strains might result from fungal auxin production. We thus measured auxin production by the two *Trichoderma* strains in culture, in presence and absence of tryptophan, an auxin precursor. This experiment shows that the two strains can produce auxin in culture and that addition of tryptophan enhances IAA production by 2.5%, and 1.23%, for *T. guizhouense* and *T. afroharzianum* respectively.

320 These experiments show that wheat inoculation by the *Trichoderma* Lebanese strains enhances root growth and this might result from auxin production by the fungi.

322
323 **Inoculation by the *Trichoderma* Lebanese strains enhances wheat yield**

324 As shown above, *Trichoderma* inoculation by the two *Trichoderma* strains enhanced wheat seedling
325 growth. We thus tested their effect on the complete development of the wheat plants grown in soil. The
326 two inoculation methods described above were used before plant transplantation in pots but no
327 significant difference could be observed as a result of the inoculation method. Plants were grown to
328 maturity and the different wheat yield components measured (Figure 5, Supplementary Figure 2). This
329 analysis showed that the plant weight (Figure 5A), the number of spikes per plant (Figure 5B), the total
330 grain weight per plant (Figure 5C) and the grain number per plant (Figure 5D) were increased following
331 inoculation by the two fungi. In contrast the 100 grains weight remained unchanged. Altogether, yield
332 harvest index (HI%) was increased around 6% (PDA inoculation method) and 10% (spore inoculation
333 method) by the fungal inoculations (Supplementary Figure 2B).

334 In order to know whether fungal inoculation could change seed protein quantity as well as seed protein
335 pattern of the grains we analyzed them in plants inoculated by the two strains and following the two
336 inoculation methods. As shown on Supplementary Figure 2C neither the protein contents nor the
337 protein profiles were modified in the wheat grains by the *Trichoderma* inoculation.

338 Thus, inoculation of plants, independently of the method used increased wheat yield including number
339 of spikes/plants, number of grains/plants, grain weight/plant, whole plant dry weight, and harvesting
340 index (HI%), but did not modify the seed protein content

341

342 **Discussion**

343 Plants interact with many microorganisms in nature and it is believed that a better understanding of
344 these interactions can help develop a sustainable agriculture less dependent from pesticides and
345 polluting fertilizers. Among interacting microorganisms some are deleterious (pathogens) but others
346 are beneficial (Singh et al., 2018). The fungus *Trichoderma* is widely studied as a beneficial
347 microorganism, promoting plant growth but also protecting plants against pathogens (Mukhopadhyay
348 & Kumar, 2020). *Trichoderma* can be used as PGPF with dicot or monocot crops like tomato and wheat
349 for example (Sood et al., 2020; Stewart & Hill, 2014).

350 The beneficial natural endophyte characteristic of most of the *T. harzianum* species members, on a
351 wide number of plant species, resulted in their used as bio stimulants improving plant development,
352 nutrient absorption, as well as resistance to various biological and environmental stresses (Kakabouki
353 et al., 2021). During endophytic interaction, *Trichoderma* colonizes the root surface and produces
354 various changes in plant morphological traits when the hyphae begin to penetrate the root epidermis.
355 The production of hydrolytic enzymes such as cellulases and proteases typically aids root penetration
356 (Enshasy et al., 2020) but also prime the plant defenses, enhancing protection against pathogens
357 (Mukhopadhyay & Kumar, 2020). *Trichoderma* spp. can also produce elicitors that may manipulate
358 hormone signal transduction, including salicylic acid (SA), jasmonic acid (JA), and ethylene (ET),
359 frequently involved in systemic resistance in plants (Nawrocka & Małolepsza, 2013; Zhao et al., 2021).
360 *Trichoderma* species can also improve plant growth through a variety of other mechanisms, including
361 mycoparasitism, antibiosis, toxin degradation, inactivation of pathogenic enzyme pathways, resistance
362 against pathogens, enhanced nutrient solubilization and uptake, inorganic nutrient sequestration,
363 together with enhanced root hair development (Shahriar et al., 2022).

364 Among crops, wheat is one of the major staple foods around the world. Previous studies have shown
365 that it can benefit from interaction with *Trichoderma* fungi for growth promotion or pathogen
366 protection (TariqJaveed et al., 2021). In this study we have isolated two *Trichoderma* strains from
367 Lebanese soils, characterized them, and evaluated their growth promotion effect on wheat. Molecular
368 and taxonomic characterization showed that the two strains are *T. guizhouense*, and *T. afroharzianum*
369 isolates belonging to the Harzianum complex (Chaverri et al., 2015; Stummer et al., 2020). The
370 morphology of the two strains is in agreement with *Trichoderma* morphology (Zhu & Zhuang, 2015).
371 In addition, these two isolates were also able to colonize the root rhizoderm, and external cortex, while
372 markedly enhancing seedling root growth. In contrast our study suggested that the two Lebanese
373 isolates lack the ability to colonize wheat aerial tissues under the conditions used. Growth conditions
374 might influence colonization as the *T. afroharzianum* strain was isolated from the base of the aerial
375 part of a field grown plant.

376 The influence of the two *Trichoderma* strains inoculation on wheat root development suggested that
377 they may produce auxin (Nieto-Jacobo et al., 2017). IAA is the main auxin promoting root system
378 development, and able to modify root architecture in the *Trichoderma* plant interaction (Illescas et al.,
379 2021). Additional *Trichoderma* metabolites, and proteins (effectors) are also known to significantly
380 modulate IAA production in plants, resulting in root hair growth and enhanced root mass development
381 (Illescas et al., 2021). These results are in agreement with previous studies showing that auxin-
382 generating *Trichoderma* strains interact with wheat and other plants (Illescas et al., 2021; Pelagio-
383 Flores et al., 2017). After root colonization, *Trichoderma* fungi stimulate root development and modify
384

385

386

plant metabolism, by releasing numerous secondary compounds (Kakabouki et al., 2021). For example, harzianic acid is one of *T. harzianum* metabolites that is responsible for promoting plant growth (Vinale et al., 2014; Xie et al., 2021) and acting as biocontrol agent.

387

388

389

390

391

392

393

Recent studies carried out on model plants like *Arabidopsis thaliana* inoculated by *T. atroviride*, and *T. guizhouense* strains showed that *Trichoderma* volatiles increased endogenous sugar contents in shoots, roots and root exudates, and could enhance *Arabidopsis* root development, root architecture, and promote the symbiosis efficiency. These effects were suggested to be related to auxin transport and signaling (Esparza-Reynoso et al., 2021; Y. Li et al., 2022). In the meantime, approaches of inoculating *Trichoderma* to promote plant growth varied between seed/seedling treatments, soil processing, and foliar spray (Bhandari et al., 2021).

394

395

396

397

398

399

IAA biosynthesis in fungi is tryptophan-dependent, and supplying this amino acid to the medium, increases IAA production (Nieto-Jacobo et al., 2017). We indeed could enhance IAA production in culture of *T. afroharzianum*, and *T. guizhouense* by adding tryptophan to the medium. Further studies have to be carried out to indicate the optimum IAA concentration, required to reach the maximum root growth, and/or yield, and good agronomic traits, with precise determination of *Trichoderma* inoculums (Harman, 2000).

400

401

402

403

404

405

In this work we tested the ability of *T. afroharzianum* and *T. guizhouense* to enhance wheat growth (plant growth promotion) using two seed inoculation methods: 1) PDA mediated inoculation, and 2) spore inoculation. Our resulted showed a notable increased in seedling development during the first 10 days following seed inoculation by the *T. afroharzianum*, and *T. guizhouense* fungi (Figure 4. A,B). This result is in agreement with previous studies (Kucuk, 2014) showing that root and shoot growth of wheat could be improved by *T. harzianum*, isolated from Turkey.

406

407

408

409

410

411

412

413

414

Similar effects were observed when inoculating seedlings with other *Trichoderma* spp. (Oliveira *et al.* 2018; Kthiri *et al.* 2020; Mahato, Bhuju, et Shrestha 2018; Anjum *et al.* 2020). These studies indicated that many growth or yield parameters like the percentage of germination (PG), root length (RL), shoot length (SL), total length (TL), fresh root mass (FRM), fresh shoot mass (FSM), total fresh mass (TFM), dry root mass (DRM), dry shoot mass (DSM), total biomass (BIO), root mass ratio (RMR), shoot mass ratio (SMR) and aerial part/root system ratio (AP/RS), were augmented. Similarly, *T. yunnanense*, and *T. afroharzianum* isolates produced IAA under salt stress (200 mM), and could stimulate photosynthesis, water utilization, and growth of wheat in saline conditions (Oljira *et al.* 2020; Zhang, Gan, et Xu 2018).
415

Our study is also in agreement with previous studies (Colla *et al.* 2015; Silletti *et al.* 2021), where seed coating could encourage seed germination, and the quality of yield and grain, including protein and mineral composition in durum wheat. Xue *et al.* (2017) inoculated six different *Trichoderma* strains on wheat seedlings and found that the six strains had the ability of increasing plant dry weight, and total yield, over a three years experiment. This is in line with our data showing that treatment with the two Lebanese *Trichoderma* strains could significantly affect plant yield. The HI% indicated an increased yield, as a response to treatments with both *T. guizhouense* and *T. afroharzianum* strains but *T. afroharzianum* showed higher impact on yield and its components. The fungal growth promotion effects are not completely described but it is known that *Trichoderma* can stimulate plant food scanning by enhancing root elongation in order to invade more, either shallow surface, or even deeper underneath areas, seeking for nutrients uptake. This capacity probably essentially relies on auxin production (Zin & Badaluddin, 2020). In agreement with this, in our study *T. afroharzianum* and *T. guizhouense* isolated strains effectively strongly promoted primary and secondary root elongation.

In addition, *Trichoderma spp.* are known to increase the plant nutrient availability needs by solubilizing minerals such as Fe, Mn, Zn, and P, and providing extra nitrogen to the plant. As a result, after *Trichoderma* colonization, secondary metabolites production is also increased (Kakabouki et al., 2021).

Protein content of grains is an important agronomic character for wheat (Illescas et al., 2022). Protein analysis of the grains revealed that in our experiments the inoculation by *Trichoderma spp.* did not modify their protein nature and content. This shows that *Trichoderma* can promote wheat growth and enhance grain production, without changing the grain protein composition. This observation might be correlated with the other part of our study indicating that the weight of hundred grains is not changed despite yield increase.

In conclusion, with the increased global demand, during a chain of serious crisis, and environmental impacts, expanding wheat production is considered an urgent request. Unconventional strategies are necessary for guaranteeing food security and providing sustainability with safer applications. In this study two *Trichoderma* strains were isolated from the Lebanese lands, identified, characterized, and proved to be endophytes. We proposed that IAA production by the two strains may explain their growth promoting effect.

445
446 **Conflict of Interest**

447 The authors declare that the research was conducted in the absence of any commercial or financial

448 relationships that could be construed as a potential conflict of interest.

449 **Author Contributions**

450 NM, PR, MD, FA and NB conceived and designed the experiments. NM, CM, PR, MD, GA, AP, BC

451 and LR conducted the experiments and analyses. PR, NM, GA, and FA drafted the manuscript. All

452 authors have read and approved the final version of the manuscript.

453

454

455 **Funding**

456 This work was funded by the ‘AUF-CNRSL-UL’, Lebanon Francophonie University Agency (AUF),

457 the National Council for Scientific Research in Lebanon (CNRS-L) and the Lebanese University (UL).

458 This work was also supported by the CNRS and has benefited from the support of the LabEx Saclay

459 Plant Sciences (ANR-10-LABX-0040-SPS, LabEx SPS and ANR-17-EUR-0007, EUR SPS-GSR)

460 which is managed by the French National Research Agency under the program ‘Investissements

461 d'avenir' (ANR-11-IDEX-0003-02).

462

463 **Acknowledgments**

464 Authors would like to thank ICARDA and LARI for supporting this research by providing samples

465 and materials.

466

467

468 **References**

469 Alfano, G., Ivey, M. L. L., Cakir, C., Bos, J. I. B., Miller, S. A., Madden, L. V., Kamoun, S., and

470 Hoitink, H. a. J. (2007). Systemic Modulation of Gene Expression in Tomato by *Trichoderma*

471 *hamatum* 382. *Phytopathology*®, 97(4), 429-437. doi: 10.1094/PHYTO-97-4-0429

472 Almi, H., and Dehimat, L. (2016). Etude des myco-pathogènes de Lens culinaris et évaluation de l’effet

473 de deux souches de *Trichoderma harzianum*. http://depot.umc.edu.dz/handle/123456789/5076

474 ASCAME. (2022). *COVID-19 and the Russian invasion of Ukraine*.
475 Asseng, S., Foster, I., and Turner, N. C. (2011). The impact of temperature variability on wheat yields.
476 *Global Change Biology*, 17(2), 997-1012. doi: 10.1111/j.1365-2486.2010.02262.x

477 Atoui, A., El Khoury, A., Kallassy, M., and Lebrihi, A. (2012). Quantification of *Fusarium graminearum* and *Fusarium culmorum* by real-time PCR system and zearalenone assessment in maize. *International Journal of Food Microbiology*, 154(1-2), 59-65. doi: 10.1016/j.ijfoodmicro.2011.12.022

481 Bhandari, S., Pandey, K. R., Joshi, Y., and Lamichhane, S. (2021). An overview of multifaceted role of *Trichoderma spp*. For sustainable agriculture. doi: 10.26832/24566632.2021.0601010

483 Brotman, Y., Lisec, J., Méret, M., Chet, I., Willmitzer, L., and Viterbo, A. (2012). Transcript and metabolite analysis of the Trichoderma-induced systemic resistance response to *Pseudomonas syringae* in Arabidopsis thaliana. *Microbiology (Reading, England)*, 158(Pt 1), 139-146. doi: 10.1099/mic.0.052621-0

487 Carro-Huerga, G., Mayo-Prieto, S., Rodríguez-González, Á., Álvarez-García, S., Gutiérrez, S., and Casquero, P. A. (2021). The Influence of Temperature on the Growth, Sporulation, Colonization, and Survival of *Trichoderma spp*. In *Grapevine Pruning Wounds*. *Agronomy*, 11(9), 1771. doi: 10.3390/agronomy11091771

491 Chaverri, P., Branco-Rocha, F., Jaklitsch, W., Gazis, R., Degenkolb, T., and Samuels, G. J. (2015). Systematics of the *Trichoderma harzianum* species complex and the re-identification of commercial biocontrol strains. *Mycologia*, 107(3), 558-590. doi: 10.3852/14-147

494 Colla, G., Rouphael, J., Bonini, P., and Cardarelli, M. (2015). Coating seeds with endophytic fungi enhances growth, nutrient uptake, yield and grain quality of winter wheat. *International Journal of Plant Production*, 9, 171-189.

497 Delgado-Sánchez, P., Ortega, A., Rodríguez-Hernández, A. A., Jimenez, J. F., Flores, J. (2010). Further evidence from the effect of fungi on breaking Opuntia seed dormancy. *Plant Signaling & Behavior*, 5(10), 1229-1230. doi: 10.4161/psb.5.10.12835

500 Delgado-Sánchez, P., Ortega-Amaro, M. A., Jiménez-Bremont, J. F., and Flores, J. (2011). Are fungi important for breaking seed dormancy in desert species? Experimental evidence in Opuntia streptacantha (Cactaceae). *Plant Biology* (Stuttgart, Germany), 13(1), 154-159. doi: 10.1111/j.1438-8677.2010.00333.x
504 Dennis, C., and Webster, J. (1971a). Antagonistic properties of species-groups of *Trichoderma*: I. Production of non-volatile antibiotics. *Transactions of the British Mycological Society*, 57(1), 25-IN3. doi: 10.1016/S0007-1536(71)80077-3

507 Dennis, C., and Webster, J. (1971b). Antagonistic properties of species-groups of *Trichoderma*: II. Production of volatile antibiotics. *Transactions of the British Mycological Society*, 57(1), 41-IN4. doi: 10.1016/S0007-1536(71)80078-5

510 Dixon, J., Braun, H. J., and Crouch, J. (2009). Transitioning wheat research to serve the future needs of the developing world. *Wheat Facts and Futures*, 1-25.

512 Enshasy, H. E. E., Ambehabati, K. K., Hanapi, S. Z., Dailin, D., Elsayed, E. A., Sukmawati, D., and Malek, R. (2020). *Trichoderma spp.: A Unique Fungal Biofactory for Healthy Plant Growth*. doi: 10.1007/978-981-15-9154-9_24

515 Esparza-Reynoso, S., Ruíz-Herrera, L. F., Pelagio-Flores, R., Macías-Rodríguez, L. I., Martínez-Trujillo, M., López-Coria, M., Sánchez-Nieto, S., Herrera-Estrella, A., and López-Bucio, J. (2021). *Trichoderma atroviride*-emitted volatiles improve growth of Arabidopsis seedlings through modulation of sucrose transport and metabolism. *Plant, Cell & Environment*, 44(6), 1961-1976. doi: 10.1111/pce.14014

520 Esposito, E., and da Silva, M. (1998). Systematics and Environmental Application of the Genus *Trichoderma*. *Critical Reviews in Microbiology*, 24(2):89-98. doi: 10.1080/10408419891294190

523 Felsenstein, J. (1985). Confidence Limits on Phylogenies: An Approach Using the Bootstrap. *Evolution*, 39(4), 783-791. doi: 10.2307/2408678

525 Ghazanfar, M., Raza, M., Raza, W., and Qamar, M. (2018). *TRICHODERMA AS POTENTIAL BIOCONTROL AGENT, ITS EXPLOITATION IN AGRICULTURE*: A REVIEW. 2, 2617-1279.

528 Glauben, T., Svanidze, M., Götz, L., Prehn, S., Jamali Jaghdani, T., Djuric, I., and Kuhn, L. (2022). The War in Ukraine, Agricultural Trade and Risks to Global Food Security. *Intereconomics*, 57, 157-163. doi: 10.1007/s10272-022-1052-7

531 Gusmiaty, Restu, A. M., and Payangan, R. Y. (2019). Production of IAA (Indole Acetic Acid) of the rhizosphere fungus in the Suren community forest stand. *343*(1), 012058. doi: 10.1088/1755-1315/343/1/012058
534

Harman, G. E. (2000). Myths and Dogmas of Biocontrol Changes in Perceptions Derived from

535

Research on *Trichoderma harzianum* T-22. *Plant Disease*, 84(4), 377-393. doi:

536

10.1094/PDIS.2000.84.4.377

537

Harman, G. E., and Kubicek, C. P. (2002). *Trichoderma And Gliocladium*. Volume 1 : Basic Biology,

538

Taxonomy and Genetics. CRC Press.

539

Illescas, M., Morán-Diez, M. E., Martínez de Alba, Á. E., Hermosa, R., and Monte, E. (2022). Effect

540

of *Trichoderma asperellum* on Wheat Plants’ Biochemical and Molecular Responses, and Yield

541

under Different Water Stress Conditions. *International Journal of Molecular Sciences*, 23(12),

542

6782. doi: 10.3390/ijms23126782

543

Illescas, M., Pedrero-Méndez, A., Pitorini-Bovolini, M., Hermosa, R., and Monte, E. (2021).

544

Phytohormone Production Profiles in *Trichoderma* Species and Their Relationship to Wheat

545

Plant Responses to Water Stress. *Pathogens*, 10(8), 991. doi: 10.3390/pathogens10080991

546

Jaber, L. R. (2018). Seed inoculation with endophytic fungal entomopathogens promotes plant growth

547

and reduces crown and root rot (CRR) caused by *Fusarium culmorum* in wheat. *Planta*, 248(6),

548

1525-1535. doi: 10.1007/s00425-018-2991-x

549

Kakabouki, I., Tataridas, A., Mavroeidis, A., Kousta, A., Karydogianni, S., Zisi, C., Kouneli, V.,

550

Konstantinou, A., Folina, A., Konstantas, A., and Papastylianou, P. (2021). Effect of

551

Colonization of *Trichoderma harzianum* on Growth Development and CBD Content of Hemp

552

(*Cannabis sativa* L.). *Microorganisms*, 9(518), 518. doi: 10.3390/microorganisms9030518

553

Kapusi, E., and Stoger, E. (2022). Molecular Farming in *SeedSeedsCrops* : Gene TransferGenetransfer

554

into BarleyBarley (Hordeum vulgareHordeum vulgare) and WheatWheat (*Triticum*

555

aestivumTriticum aestivum). In S. Schillberg & H. Spiegel (Éds.), *Recombinant Proteins in*

556

*Plants: Methods and Protocols* (p. 49-60). Springer US. doi: 10.1007/978-1-0716-2241-4_3

557

Kthiri, Z., Jabeur, M. B., Machraoui, M., Gargouri, S., Hiba, K., and Hamada, W. (2020). Coating

558

seeds with *Trichoderma* strains promotes plant growth and enhance the systemic resistance

559

against *Fusarium* crown rot in durum wheat. doi: 10.1186/s41938-020-00338-6

560

Kubicek, C. P., Bissett, J., Druzhinina, I., Kullnig-Gradinger, C., and Szakacs, G. (2003). Genetic and

561

metabolic diversity of *Trichoderma*: A case study on South-East Asian isolates. *Fungal*

562

*Genetics and Biology: FG & B*, 38(3), 310-319. doi: 10.1016/s1087-1845(02)00583-2
563

Kucuk, C. (2014). Enhanced root and shoot growth of wheat (Triticum aestivum L.) by *Trichoderma harzianum* from Turkey. *Pakistan journal of biological sciences : PJBS*.

564

565

Kumar, S., Stecher, G., Li, M., Knyaz, C., and Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. *Molecular Biology and Evolution, 35*(6), 1547-1549. doi: 10.1093/molbev/msy096

566

567

568

Kuznetsova, A., Brockhoff, P., and Christensen, R. (2017). *lmerTest Package : Tests in Linear Mixed Effects Models*. doi: 10.18637/JSS.V082.I13

569

570

Li, G., Boontung, R., Powers, C., Belamkar, V., Huang, T., Miao, F., Baenziger, P. S., and Yan, L. (2017). Genetic basis of the very short life cycle of « *Apogee* » wheat. *BMC Genomics*, 18(1), 838. doi: 10.1186/s12864-017-4239-8

571

572

573

Li, Y., Shao, J., Fu, Y., Chen, Y., Wang, H., Xu, Z., Feng, H., Xun, W., Liu, Y., Zhang, N., Shen, Q., Xuan, W., and Zhang, R. (2022). The volatile cedrene from *Trichoderma guizhouense* modulates Arabidopsis root development through auxin transport and signalling. https://pubag.nal.usda.gov/catalog/7687078

574

575

576

577

Mastouri, F., Björkman, T., and Harman, G. (2010). Seed Treatment with *Trichoderma harzianum* Alleviates Biotic, Abiotic, and Physiological Stresses in Germinating Seeds and Seedlings. *Phytopathology*, 100, 1213-1221. doi: 10.1094/PHYTO-03-10-0091

578

579

580

Mastouri, F., Björkman, T., and Harman, G. E. (2012). *Trichoderma harzianum* enhances antioxidant defense of tomato seedlings and resistance to water deficit. *Molecular Plant-Microbe Interactions: MPMI*, 25(9), 1264-1271. doi: 10.1094/MPMI-09-11-0240

581

582

583

Morán-Diez, E., Rubio, B., Domínguez, S., Hermosa, R., Monte, E., and Nicolás, C. (2012). Transcriptomic response of Arabidopsis thaliana after 24 h incubation with the biocontrol fungus *Trichoderma harzianum*. *Journal of Plant Physiology*, 169(6), 614-620. doi: 10.1016/j.jplph.2011.12.016

584

585

586

587

Mukhopadhyay, R., and Kumar, D. (2020). *Trichoderma*: A beneficial antifungal agent and insights into its mechanism of biocontrol potential. *Egyptian Journal of Biological Pest Control*, 30(1), 133. doi: 10.1186/s41938-020-00333-x

588

589

590

Nawrocka, J., and Małolepsza, U. (2013). Diversity in plant systemic resistance induced by *Trichoderma*. doi: 10.1016/J.BIOCONTROL.2013.07.005

591
592 Nieto-Jacobo, M. F., Steyaert, J. M., Salazar-Badillo, F. B., Nguyen, D. V., Rostás, M., Braithwaite, M., De Souza, J. T., Jimenez-Bremont, J. F., Ohkura, M., Stewart, A., and Mendoza-Mendoza, A. (2017). Environmental Growth Conditions of *Trichoderma* spp. Affects Indole Acetic Acid Derivatives, Volatile Organic Compounds, and Plant Growth Promotion. *Frontiers in Plant Science*, 8, 102. doi: 10.3389/fpls.2017.00102

597 Oliveira, J. B. D., Muniz, P. H. P. C., Peixoto, G. H. S., Oliveira, T. A. S. D., Duarte, E. A. A., 598 Rodrigues, F., and Carvalho, D. D. C. (2018). Promotion of Seedling Growth and Production 599 of Wheat by Using *Trichoderma* spp. *Journal of Agricultural Science*, 10(8), undefined- 600 undefined. doi: 10.5539/jas.v10n8p267

601 Oljira, A. M., Hussain, T., Waghmode, T., Zhao, H., Sun, H., Liu, X., Wang, X., and Liu, B. (2020). 602 Trichoderma Enhances Net Photosynthesis, Water Use Efficiency, and Growth of Wheat 603 (*Triticum aestivum* L.) under Salt Stress. *Microorganisms*, 8, 1565. doi: 604 10.3390/microorganisms8101565

605 Pelagio-Flores, R., Esparza-Reynoso, S., Garnica-Vergara, A., López-Bucio, J., and Herrera-Estrella, 606 A. (2017). Trichoderma-Induced Acidification Is an Early Trigger for Changes in Arabidopsis 607 Root Growth and Determines Fungal Phytostimulation. *Frontiers in Plant Science*, 8. 608 https://www.frontiersin.org/articles/10.3389/fpls.2017.00822

609 Shahriar, S. A., Islam, M. N., Chun, C. N. W., Kaur, P., Rahim, M. A., Islam, M. M., Uddain, J., and 610 Siddiquee, S. (2022). Microbial Metabolomics Interaction and Ecological Challenges of 611 *Trichoderma* Species as Biocontrol Inoculant in Crop Rhizosphere. *Agronomy (Basel)*, 12(4). 612 doi: 10.3390/agronomy12040900

613 Shoresh, M., Harman, G. E., and Mastouri, F. (2010). Induced systemic resistance and plant responses 614 to fungal biocontrol agents. *Annual Review of Phytopathology*, 48, 21-43. doi: 615 10.1146/annurev-phyto-073009-114450

616 Silletti, S., Di Stasio, E., Van Oosten, M. J., Ventorino, V., Pepe, O., Napolitano, M., Marra, R., Woo, 617 S. L., Cirillo, V., and Maggio, A. (2021). Biostimulant Activity of *Azotobacter chroococcum* 618 and *Trichoderma harzianum* in Durum Wheat under Water and Nitrogen Deficiency. 619 *Agronomy*, 11(2), 380. doi: 10.3390/agronomy11020380

620 Singh, A., Shukla, N., Kabadwal, B. C., Tewari, A., and Kumar, J. (2018). Review on Plant- 621 *Trichoderma*-Pathogen Interaction. *International Journal of Current Microbiology and 622 Applied Sciences*, 7, 2382-2397. doi: 10.20546/ijcmas.2018.702.291
Sood, M., Kapoor, D., Kumar, V., Sheteiwy, M. S., Ramakrishnan, M., Landi, M., Araniti, F., and Sharma, A. (2020). *Trichoderma*: The “Secrets” of a Multitalented Biocontrol Agent. *Plants*, 9(6), 762. doi: 10.3390/plants9060762

Stewart, A., and Hill, R. (2014). Applications of *Trichoderma* in Plant Growth Promotion. In *Biotechnology and Biology of Trichoderma* (p. 415-428). doi: 10.1016/B978-0-444-59576-8.00031-X

Stummer, B., Zhang, Q., Zhang, X., Warren, R., and Harvey, P. (2020). Quantification of *Trichoderma afroharzianum*, *Trichoderma harzianum* and *Trichoderma gamsii* inoculants in soil, the wheat rhizosphere and in planta suppression of the crown rot pathogen *Fusarium pseudograminearum*. *Journal of Applied Microbiology*, 129. doi: 10.1111/jam.14670

Tamura, K., and Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. *Molecular Biology and Evolution*, 10(3), 512-526. doi: 10.1093/oxfordjournals.molbev.a040023

TariqJaveed, M., Farooq, T., Al-Hazmi, A. S., Hussain, M. D., and Rehman, A. U. (2021). Role of *Trichoderma* as a biocontrol agent (BCA) of phytoparasitic nematodes and plant growth inducer. *Journal of Invertebrate Pathology*, 183, 107626. doi: 10.1016/j.jip.2021.107626

Topolovec-Pintarić, S. (2019). <em>Trichoderma</em>: Invisible Partner for Visible Impact on Agriculture. *Trichoderma - The Most Widely Used Fungicide*. doi: 10.5772/intechopen.83363

Tseng, Y.-H., Rouina, H., Groten, K., Rajani, P., Furch, A. C. U., Reichelt, M., Baldwin, I. T., Nataraja, K. N., Uma Shaanker, R., and Oelmüller, R. (2020). An Endophytic *Trichoderma* Strain Promotes Growth of Its Hosts and Defends Against Pathogen Attack. *Frontiers in Plant Science*, 11. https://www.frontiersin.org/articles/10.3389/fpls.2020.573670

Vargas, W. A., Crutcher, F. K., and Kenerley, C. M. (2011). Functional characterization of a plant-like sucrose transporter from the beneficial fungus *Trichoderma virens*. Regulation of the symbiotic association with plants by sucrose metabolism inside the fungal cells. *New Phytologist*, 189(3), 777-789. doi: 10.1111/j.1469-8137.2010.03517.x

Vargas, W. A., Mandawe, J. C., and Kenerley, C. M. (2009). Plant-derived sucrose is a key element in the symbiotic association between *Trichoderma virens* and maize plants. *Plant Physiology*, 151(2), 792-808. doi: 10.1104/pp.109.141291
652 Vinale, F., Manganiello, G., Nigro, M., Mazzei, P., Piccolo, A., Pascale, A., Ruocco, M., Marra, R.,
653 Lombardi, N., Lanzuise, S., Varlese, R., Cavallo, P., Lorito, M., and Woo, S. (2014). A Novel
654 Fungal Metabolite with Beneficial Properties for Agricultural Applications. *Molecules*. doi:
655 10.3390/molecules19079760

656 Wickham, H. (2009). Getting started with qplot. In H. Wickham (Éd.), *Ggplot2: Elegant Graphics for
657 Data Analysis* (p. 9-26). Springer. doi: 10.1007/978-0-387-98141-3_2

658 Widden, P., and Abitbol, J.-J. (1980). Seasonality of *Trichoderma* Species in a Spruce-Forest Soil.
659 *Mycologia*, 72(4), 775-784. doi: 10.1080/00275514.1980.12021246

660 Xie, L., Zang, X., Cheng, W., Zhang, Z., Zhou, J., Chen, M., and Tang, Y. (2021). Harzianic Acid from
661 *Trichoderma afroharzianum* Is a Natural Product Inhibitor of Acetohydroxyacid Synthase.
662 *Journal of the American Chemical Society*. doi: 10.1021/jacs.1c03988

663 Xue, A., Guo, W., Chen, Y., Siddiqui, I., Marchand, G., Liu, J., and Ren, C. (2017). Effect of seed
664 treatment with novel strains of *Trichoderma spp*. On establishment and yield of spring wheat.
665 doi: 10.1016/J.CROPRO.2017.02.003

666 Zhao, J., Liu, T., Liu, W., Zhang, D., Dong, D., Wu, H., Zhang, T., and Liu, D. (2021). Transcriptomic
667 insights into growth promotion effect of *Trichoderma afroharzianum* TM2-4 microbial agent
668 on tomato plants. *Journal of Integrative Agriculture*, 20(5), 1266-1276.
669 doi.org/10.1016/S2095-3119(20)63415-3

670 Zhu, Z., and Zhuang, W. (2015). *Trichoderma* (Hypocrea) species with green ascospores from China.
671 *Persoonia*. doi: 10.3767/003158515X686732

672 Zin, N. A., and Badaluddin, N. A. (2020). Biological functions of *Trichoderma spp*. For agriculture
673 applications. doi: 10.1016/J.AOAS.2020.09.003

674

675

676 **Figure legends**

677 **Figure 1.** Phylogenetic tree of CAL-EF-ITS branch includes only species in the *T. harzianum* complex.

678 Two species are identified from Lebanon and are placed in this tree: *T. afroharzianum* (T. S3PA) and

679 *T. guizhouense* (T. S3SB). Strains used in this phylogenetic analysis are described in supplementary
Table 1 with their corresponding geographic origin, substrate, voucher number and GenBank accession numbers. *T. agressivum* is used as an outgroup in this analysis.

**Figure 2.** Growth of *T. guizhouense* (A) and *T. afroharzianum* (B) for one week on PDA supplemented with glucose. The age of the culture is indicated below the picture in hours.

Microscopic visualization of conidiophores of *T. guizhouense* (C) *T. afroharzianum* (D) (x1000). (I) hyphae, (II) conidiophores, (III) Phialides and (IV) conidia (the scale bar corresponds to 10µm).

**Figure 3.** A-D: cross section of wheat root seedlings inoculated with *T. guizhouense* (A, B) or *T. afroharzianum* (C, D) using the PDA inoculation method. E-H: cross section of wheat root seedlings inoculated with *T. guizhouense* (E, F), with *T. afroharzianum* (G, H) using the spore inoculation method. (Ep) epidermis, (C) cortex (Cc) central cylinder, (Hy) fungal hyphae. The fungal hyphae are stained green using WGA-Alexa Fluor 488, and the root cell wall are stained red using propidium iodide, (BDFH correspond to the boxes of ACEG respectively).

**Figure 4.** Growth of roots and aerial parts after 10 days of culture, with and without *Trichoderma* (A); I) not inoculated, II) a representative seedling inoculated with *T. guizhouense*, III) a representative seedling inoculated with *T. afroharzianum*, (B) Total root, and aerial length (cm) of non-inoculated (N.I.) or inoculated wheat plants with *T. guizhouense* (T.G.) and *T. afroharzianum* (T.A.), respectively, (C) IAA production capability of the two *Trichoderma* isolates (T.G. and T.A.) with and without tryptophan.

**Figure 5.** Yield parameters: A) Total dry plant weight (g)/plant; B) Number of spikes/plant; C) Total grains weight (g)/plant; D) Number of grains/plant, as influenced by PDA and spore inoculation methods. N.I.: non-inoculated; T.G.: wheat plants inoculated with *T. guizhouense*; T.A.: wheat plants inoculated with *T. afroharzianum*. Values in the same graph having different letters are significantly different, at P≤0.01 (ANOVA test).
708 **Supplementary material**

709 **Supplementary Table 1:** Strains used in the phylogenetic analyses, with their corresponding
710 geographic origin, substrate, voucher number and GenBank accession numbers

711 **Supplementary Table 2:** Fungal strains isolated from Lebanon from different ecological site.

712 **Supplementary Figure 1.** (A) principal root length (cm) over a 10 days period. (B) total root, and
713 aerial length (cm). (C) total root, and aerial mass (g). N.I.: non-inoculated; T.G.: wheat plants
714 inoculated with *T. guizhouense*; T.A.: wheat plants inoculated with *T. afroharzianum*. Values in the
715 same column having different letters are significantly different, at P≤0.01 (ANOVA test).

716 **Supplementary Figure 2.** (A) Harvest index (%) and (B) 100 grain weight (g). N.I.: non-inoculated;
717 T.G.: wheat plants inoculated with *T. guizhouense*; T.A.: wheat plants inoculated with *T.
718 afroharzianum*. (C) protein analysis (composition and content) of wheat plants non-inoculated (cont
719 or inoculated with *T. guizhouense* (TGUI) or *T. afroharzianum* (Taf) as influenced by PDA
720 inoculation method (PDA) or the spore inoculation method (Tubes). The protein content of 100
721 grains in each condition is indicated below the gel pictures. Values in each graph having different
722 letters are significantly different, at P≤0.01 (ANOVA test).

723
## Supplementary Material

### Supplementary Tables

**Supplementary Table 1.** Strains used in the phylogenetic analyses, with their corresponding geographic origin, substrate, voucher number and GenBank accession numbers

<table><thead><tr><th rowspan="2">Species</th><th rowspan="2">Voucher/culture Nos.</th><th rowspan="2">Geographic origin</th><th rowspan="2">Substrate</th><th colspan="3">Genbank accession Nos.</th></tr><tr><th>CAL</th><th>ITS</th><th>EF1</th></tr></thead><tbody><tr><td><i>T. afarasin</i></td><td>CBS 130742<br>= Dis 314f</td><td>Cameroon</td><td><i>Cola altissima</i><br>trunk endophyte</td><td>FJ442312</td><td>FJ442259</td><td>FJ463400</td></tr><tr><td><i>T. afarasin</i></td><td>Dis 377a</td><td>Cameroon</td><td><i>Cola</i> sp. trunk<br>endophyte</td><td>KP115277</td><td>FJ442665</td><td>FJ463322</td></tr><tr><td><i>T. afarasin</i></td><td>CBS 130501<br>= G.J.S. 06-98</td><td>Cameroon</td><td>Soil</td><td>FJ442353</td><td>FJ442630</td><td>FJ463327</td></tr><tr><td><i>T. afarasin</i>*</td><td>CBS 130755<br>= IMI 393967</td><td>Cameroon</td><td>Soil</td><td>FJ442388</td><td>AY027784</td><td>AF348093</td></tr></tbody></table>
= G.J.S. 99-227

T. G. Harman AF469190 AF469188 AF469194
**afroharzianum** 1295-22 and New York² G. Harman, patented biocontrol strain
= ATCC 29847
= T22
= GJS 94-26

T. G.J.S. 00-24 Mexico Soil AF442880 AF443922 AF443940
**afroharzianum**

T. CBS 124620 Peru On basidioma of *Moniliophthora roreri* on fruit of *Theobroma*

T. CBS 124614 Peru On basidioma of *Moniliophthora roreri* on fruit of *Theobroma*

T. G.J.S. 05-113 Italy Wheat seed FJ442371 FJ442235 FJ463378
**afroharzianum**

T. G.J.S. 97-263 Japan Soil AF442877 AF194010 AF348091
**afroharzianum**
<table>
  <tr>
    <td>T. afroharzianum</td>
    <td>G.J.S. 97-268</td>
    <td>Japan</td>
    <td>Soil</td>
    <td>AF442878</td>
    <td>AF194015</td>
    <td>AF348105</td>
  </tr>
  <tr>
    <td>T. afroharzianum</td>
    <td>IMI 393972</td>
    <td>Cameroon</td>
    <td>Soil</td>
    <td>AF442882</td>
    <td>AY027781</td>
    <td>AF348106</td>
  </tr>
  <tr>
    <td>T. aggressivum</td>
    <td>CBS 433.95</td>
    <td>Northern Ireland</td>
    <td>Mushroom compost</td>
    <td>FJ442279</td>
    <td>FJ442605</td>
    <td>AF348097</td>
  </tr>
  <tr>
    <td>T. atrobrunneum</td>
    <td>CBS 130440</td>
    <td>Italy</td>
    <td>Soil</td>
    <td>FJ442329</td>
    <td>FJ442273</td>
    <td>FJ463360</td>
  </tr>
  <tr>
    <td>T. atrobrunneum</td>
    <td>G.J.S. 04-67</td>
    <td>Italy</td>
    <td>Castanea sativa twig</td>
    <td>FJ442364</td>
    <td>FJ442234</td>
    <td>FJ463299</td>
  </tr>
  <tr>
    <td>T. atrobrunneum</td>
    <td>G.J.S. 05-100</td>
    <td>Italy</td>
    <td>Soil</td>
    <td>FJ442331</td>
    <td>FJ442677</td>
    <td>FJ463392</td>
  </tr>
  <tr>
    <td>T. camerunense*</td>
    <td>CBS 137272</td>
    <td>Cameroon</td>
    <td>Soil</td>
    <td>AF442875</td>
    <td>AY027780</td>
    <td>AF348107</td>
  </tr>
  <tr>
    <td>T. camerunense</td>
    <td>G.J.S. 99-230</td>
    <td>Cameroon</td>
    <td>Soil</td>
    <td>AF442874</td>
    <td>AY027783</td>
    <td>AF348108</td>
  </tr>
  <tr>
    <td>T. endophyticum</td>
    <td>CBS 130730</td>
    <td>Ecuador</td>
    <td>Theobroma gileri trunk endophyte</td>
    <td>FJ442293</td>
    <td>FJ442242</td>
    <td>FJ463314</td>
  </tr>
  <tr>
    <td>T. endophyticum</td>
    <td>Dis 217h</td>
    <td>Ecuador</td>
    <td>Theobroma gileri trunk</td>
    <td>FJ442294</td>
    <td>FJ442241</td>
    <td>FJ463323</td>
  </tr>
</table>
endophyticum endophyte

T. Dis 220k Ecuador Theobroma FJ442299 FJ442270 FJ463328
endophyticum gileri trunk
endophyte

T. guizhouense DAOM 231435 Rwanda Soil FJ577721 EF191296 EF191321

T. guizhouense G.J.S. 07-18 Ghana Soil FJ442355 FJ442641 FJ463390

T. guizhouense ATCC 90179 Indonesia Dead wood AF442881 AF443923 AF443941
= IMI 374787
= G.J.S. 85-119

T. guizhouense NBRC 30608 Japan Dead branch FJ442379 DQ018116 AY937440
= IFO 30608
= G.J.S. 97-28

T. harzianum* CBS 226.95 U.K. Soil AF442864 AJ222720 AF348101
= G.J.S 95-43

T. harzianum CBS 227.95 U.K. Soil AF442866 AJ222721 AF348100
= G.J.S 95-40

T. harzianum IMI 359823 Northern
Ireland Mushroom
compost AF442865 X73690 AF348092
<table><tr><td><b><i>T. inhamatum</i></b></td><td>CBS 273.78</td><td>Colombia</td><td>Soil</td><td>AF442891</td><td>FJ442680</td><td>AF348099</td></tr><tr><td></td><td>= IMI 287526</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>= G.J.S. 95-39</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><b><i>T. lentiforme</i></b></td><td>CBS 130726</td><td>Ecuador</td><td><i>Theobroma cacao</i> trunk endophyte</td><td>FJ442287</td><td>FJ442681</td><td>FJ851872</td></tr><tr><td></td><td>= Dis 110a</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><b><i>T. lentiforme</i></b></td><td>Dis 167c</td><td>Brazil</td><td><i>Theobroma cacao</i> trunk endophyte</td><td>FJ442365</td><td>FJ442269</td><td>FJ463309</td></tr><tr><td></td><td>Dis 218e</td><td>Ecuador</td><td><i>Theobroma gileri</i> trunk endophyte</td><td>FJ442296</td><td>FJ442220</td><td>FJ463310</td></tr><tr><td><b><i>T. lixii</i>**</b></td><td>CBS 110080</td><td>Thailand</td><td>Decayed <i>Ganoderma basidiocarp</i></td><td>AF442872</td><td>AF443920</td><td>AF443938</td></tr><tr><td></td><td>= ATCC MYA-2478</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>= G.J.S. 97-96</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>= BPI 745654</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><b><i>T. neotropicale</i></b></td><td>G.J.S. 11-185</td><td>Peru</td><td><i>Hevea guianensis</i> trunk endophyte</td><td>KP115279</td><td>HQ022407</td><td>HQ022771</td></tr><tr><td></td><td>= CBS 130633</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>LA11</td><td></td><td></td><td></td><td></td><td></td></tr></table>
**T. neotropicale** G.J.S. 11-187
= T51
Peru
*Hevea brasiliensis*
trunk endophyte
KP115280
FJ884180
FJ967825

**T. rifaii** CBS 130745
= Dis 337f
Panama
*Theobroma cacao*
trunk endophyte
FJ442315
FJ442621
FJ463321

**T. simmonsii** CBS 123799
= IMI 393966
= G.J.S. 90-22
USA,
Wisconsin
Decorticated wood
AF442867
AF443915
AF443933

**T. simmonsii*** CBS 130431
= G.J.S. 91-138
USA,
Maryland
Decaying bark
AF442869
AF443917
AF443935

**T. simmonsii** CBS 546.92
= ATCC MYA-2453
= G.J.S. 92-100
USA,
Alabama
Decorticated wood
AF442871
AF443919
AF443937

**T. simmonsii** CBS 130432
= G.J.S. 94-53
BPI 749348
USA,
Illinois
Decorticated wood
AF442868
AF443916
AF443934

<footnote>* Indicates a type culture.</footnote>
** Indicates an epitype culture.

¹ CBS = CBS Fungal Biodiversity Centre culture collection, the Netherlands; DAOM. = Agriculture and Agri-Food Canada National Mycological Culture Collection; IMI. = CABI culture collection, UK; ATCC. = American Type Culture Collection, Manassas, Virginia, USA; BPI = US National Fungus Collection; WU = Herbarium WU, Institute of Botany, University of Vienna, Austria; G.J.S. = G. J. Samuels; P.C. = P. Chaverri; Hypo, C.P.K., S and WJ = W. Jaklitsch collection numbers; Dis = H.C. Evans endophyte cultures; LA, CM, PP, T = P. Chaverri endophyte cultures. Where a plant name alone is given, the substrate is twigs or branches and fungi growing on them.

² T22 is product of fusion of protoplasts of two cultures: ATCC 60850, which was isolated from soil in Colombia, and ATCC 20707, which was isolated from soil in New York state. See Ahmad and Baker (1987a,b) and Stasz et al. (1988).

Ahmad JS, Baker R. 1987a. Competitive saprophytic ability and cellulolytic activity of rhizosphere-competent mutants of *Trichoderma harzianum*. *Phytopathology* 77:358–362.

—, —. 1987b. Rhizosphere competence of *Trichoderma harzianum*. *Phytopathology* 77:182–189.

Stasz TE, Harman GE, Weeden NF. 1988. Protoplast preparation and fusion in 2 biocontrol strains of *Trichoderma harzianum*. *Mycologia* 80:141–150.

Supplementary Table 2. Fungal strains isolated from Lebanon from different ecological site.

<table><thead><tr><td>Site</td><td>Fugus location</td><td>Genus (according to ITS)</td></tr></thead><tbody></tbody></table>
**Site 1 ICARDA**

**Soil**

*   Alternaria sp. (x1)
*   Aspergillus sp. (x11)
*   Cladorrhinum sp. (x2)
*   Talaromyces sp. (x1)

**Root or stem**

*   Alternaria sp. (x2)
*   Fusarium sp. (x5)
*   Mucor sp. (x1)
*   Stromatinia sp. (x2)

**Site 2 Tal_Amara**

**Soil**

*   Aspergillus sp. (x2)
*   Penicillium sp. (x1)

**Root or stem**

*   Boeremia sp. (x1)
*   Mucor sp. (x1)

**Site 3 Doueir**

**Soil**

*   Fusarium sp. (x1)
*   Trichoderma sp. (x1)
*   Mucor sp. (x1)

**Root or stem**

*   Fusarium sp. (x3)
*   Trichoderma sp. (x1)
*   Macrophomina sp. (x1)
*   Microdochium sp. (x1)
Supplementary Figures

<figure><img src="image_2.png" /><figcaption>**Supplementary Figure 1.** (A) principal root length (cm) over a 10 days period. (B) total root, and aerial length (cm). (C) total root, and aerial mass (g). N.I.: non-inoculated; T.G.: wheat plants inoculated with *T. guizhouense*; T.A.: wheat plants inoculated with *T. afroharzianum*. Values in the same column having different letters are significantly different, at P≤0.01 (ANOVA test).</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Supplementary Figure 2.** (A) Harvest index (%) and (B) 100 grain weight (g). N.I.: non-inoculated; T.G.: wheat plants inoculated with *T. guizhouense*; T.A.: wheat plants inoculated with *T. afroharzianum*. (C) protein analysis (composition and content) of wheat plants non-inoculated (cont) or inoculated with *T. guizhouense* (TGUI) or *T. afroharzianum* (Taf) as influenced by PDA inoculation method (PDA) or the spore inoculation method (Tubes). The protein content of 100 grains in each condition is indicated below the gel pictures. Values in each graph having different letters are significantly different, at P≤0.01 (ANOVA test).</figcaption></figure>
<figure><img src="image_4.png" /></figure>

T. afroharzianum

T. atrobrunneum

T. guizhouense

T. inhamatum

T. lentiforme

T. neotropicale

T. lixii

T. afarasin

T. endophyticum

T. rifaii

T. simmonsii

T. camerunense

T. harzianum

T. aggressivum
<figure><img src="image_5.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /></figure>

A

<figure><img src="image_10.png" /></figure>

B

<figure><img src="image_11.png" /></figure>

C

<figure><img src="image_12.png" /></figure>

D

<figure><img src="image_13.png" /></figure>

E

<figure><img src="image_14.png" /></figure>

F

<figure><img src="image_15.png" /></figure>

G

<figure><img src="image_16.png" /></figure>

H
A

I

<figure><img src="image_17.png" /></figure>

II

<figure><img src="image_18.png" /></figure>

III

<figure><img src="image_19.png" /></figure>
<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>
A

<figure><img src="image_22.png" /></figure>

B

<figure><img src="image_23.png" /></figure>

C

<figure><img src="image_24.png" /></figure>

D

<figure><img src="image_25.png" /></figure>

PDA method

Spore inoculation method