First report of using Trichoderma longibrachiatum as a biocontrol agent against Macrophomina pseudophaseolina causing charcoal rot disease of lentil in Algeria

Abstract

Background

Lentil (Lens culinaris Medik.) is one of the major pulse crops in the world. The present study aimed to evaluate the effect of Trichoderma isolates to control the charcoal rot disease caused by Macrophomina pseudophaseolina recently reported on lentil plants.

Results

In this study, antagonistic effects of seven isolates of Trichoderma spp. isolated from the rhizosphere of lentil were evaluated in vitro against Macrophomina pseudophaseolina M1. All isolates of Trichoderma significantly reduced the mycelial growth of M1 in vitro. Maximum reduction of the pathogen growth was observed at T10 (72.53%), followed by T5 (63.7%) and T6 (62.88%), while T2 caused the minimum reduction (37.33%). Likewise, Trichoderma isolates significantly reduced the size and number microsclerotia. M1 produced the minimum number of microsclerotia in the presence of T10, followed by T1 causing reduction of 68.56 and 53.37% over control, respectively. The maximum reduction of microsclerotia’s size was observed in the presence of T10 (54.92%), followed by T1 (49.84) and T8 (48.68%). Results also revealed that production of volatile metabolite, components and inhibition of the test pathogen by volatile metabolites varied among different antagonistic Trichoderma. T1 produced the most volatile compounds, followed by T2 and T3 and T10 causing mycelial growth inhibition of 41.66, 33.88, 30.55 and 30.37%, respectively. The efficacy of the antagonistic biocontrol agents on charcoal rot was evaluated in vivo. Seed treatment with T10 significantly protected lentil seedlings from M. pseudophaseolina as compared to untreated plants and also improved their growth. T10 isolate was identified based on molecular tools, using internal transcribed spaces and Translation elongation factor of ribosomal DNA. The results identified T10 as Trichoderma longibrachiatum with accession number MW818102 and OK500004 deposited in NCBI GenBank database.

Conclusion

The study revealed the first report of using Trichoderma longibrachiatum as a promising biological agent against M. pseudophaseolina causing charcoal rot disease of lentil.

Background

Lentil (Lens culinaris Medik.) is one of the world’s most important and the oldest legume, having been cultivated for over 7000 years (McVicar et al. 2010). Its seeds are rich of protein and micronutrients for human and animal diets (Laskar et al. 2019). This plant has been cultivated for its capacity to fix nitrogen naturally where included in crop rotations with cereals for effective cultural control of pests, diseases and weeds, by breaking up their life cycles (Kumar 2013). In Algeria, lentil is one of the most cultivated legumes after faba bean and chickpea (FAOstat 2019). However, it is often affected by several constraints, including biotic and abiotic factors. Fungal pathogens are major factors that limit its overall yield and seeds quality such as Fusarium oxysporum f. sp. lentis causing Fusarium wilt disease, Ascochyta lentis responsible for ascochyta blight disease and Uromyces viciae-fabae, the causal agent of rust disease (Kouadri et al. 2021a).

In recent years, several new diseases were identified on lentil plants including; spot blotch and charcoal rot caused by Bipolaris sorokiniana and Macrophomina phaseolina. Macrophomina phaseolina is a fungus that incites charcoal rot disease. It has the widest host range as phytopathogenic fungus with more than 882 cultivated and wild plants including legumes and cereals (Farr and Rossman 2022). M. phaseolina is a soil and seed-borne pathogen that affects plants at all stages of development, from seedling to maturity (Purkayastha et al. 2006). The characteristic symptoms of charcoal rot are dryness of stem and roots, yellowing of leaves, and a black mass appearance of microsclerotia on stems and roots. M. phaseolina affects plants by secreting a number of cell wall degrading enzymes, including pectinases, xylanases, cellulases, and proteases (Javaid and Saddique 2012). In severe infection, plants eventually die from exposure to phaseoline toxin, along with vascular obstruction by fungal mycelium (Bhattacharya et al. 1994).

Recently, four new species of the genus Macrophomina were identified such as M. pseudophaseolina, M. euphorbicola, M. vaccini and M. tecta (Poudel et al. 2021). In 2020, M. pseudophaseolina was reported for the first time in Algeria as a new pathogen of lentil plants causing charcoal rot disease (Kouadri et al. 2021b). Control of charcoal rot disease has become difficult due to the long-term persistence of microsclerotia in soil and plant residues, pathogen's large host range, and lack of resistant cultivars; it is mainly managed by chemical fungicides (Iqbal and Mukhtar 2020a). Although chemical fungicides provide rapid and efficient protection against plant pathogens, their intensive application poses a serious threat to human health, environment and soil beneficial organisms (Aktar et al. 2009). However, the use of the same fungicides for the same pathogen leads to the development of resistant strains of the pathogens. Therefore, the researcher's efforts were focused on developing alternative eco-friendly disease control strategies, including the use of biological control agents such as Trichoderma bioagent. Trichoderma (teleomorph Hypocrea) fungi has long been used as a biocontrol agent against fungal plant pathogens (Atalla et al. 2020). Trichoderma spp. inhibit the pathogen’s activity through various mechanisms such as nutrient competition, hydrolytic enzymes, antibiotic production, plant growth promotion and defense response activation (Pal and Gardener 2006).

This study aimed to assess the efficacy of different isolates of Trichoderma as a biocontrol agent against M. pseudophaseolina in vitro and under greenhouse conditions for the control of charcoal rot disease of lentil.

Methods

Fungal material

Pathogen

In the present study, one isolate of M. pseudophaseolina M1 (MW422257, OK500004) causing charcoal rot was obtained from lentil (Lens culinaris Medik) from Relizane (Algeria) and was deposited at the phytopathological herbarium of the Ecole Nationale Supérieure Agronomique (ENSA, ex. INA, Algiers, Algeria) (Kouadri et al. 2021b). The isolate was stored on potato dextrose agar (PDA, Difco) at 4 °C. Prior to use, it was maintained and grown on potato dextrose agar (PDA) at 25 °C in darkness for 7 days.

Antagonists

Seven isolates of Trichoderma spp. were isolated from rhizosphere soils of healthy lentil plants collected from the northwest region of Algeria (Tiaret) during the period from 2019 to 2020, in order to be used as natural biocontrol agents. The samples were placed in polyethylene bags, closed tightly, and stored in a refrigerator at 4 °C until needed. Isolation of fungal antagonists was performed using a soil dilution plate method as described by Aneja (2003). One gram of dried soil samples was suspended in 9 ml sterile distilled water, agitated for 1 min. The suspension was subsequently diluted from 10⁻¹ to 10⁻⁷. The 0.1 ml soil dilutions of 10⁻³ to 10⁻⁷ were separately plated on potato dextrose agar (PDA) medium, in triplicate. The plates were then incubated at 25 °C for 3 days. After incubation, representative colonies with typical characteristics and morphological appearances of Trichoderma were selected and re-streaked on a new plate containing the same medium, to obtain pure colonies. Subsequently, the isolates were stored on PDA at 4 °C for until use.

Plant material

Lentil cultivar (Syrie 229), used in this experiment (in vivo), is a widely grown cultivar in Algeria and provided by the Algerian Office of Cereals.

Evaluation of antagonistic activity of Trichoderma isolates on mycelial growth of M. pseudophaseolina in vitro

Dual culture

In vitro inhibition of mycelial growth of M. pseudophaseolina (M1) by the Trichoderma isolates was tested using the dual culture technique described by Sivan and Chet (1989). A mycelial disc (5 mm) of 7-day-old culture from M. pseudophaseolina (M1) was placed in a Petri plate (90 mm diameter). At the opposite side of the Petri plate, a disc of Trichoderma (5 mm) of each isolate was placed separately with 3 cm of distance on a PDA medium for 5 days at 25 ± 1 °C. In control plates, the mycelial disc of test pathogen was put in the middle of the Petri plates. The experiment was repeated three times. After five days, the percentages of pathogen growth inhibition were calculated according to the following formula:

$${\text{GI }}(\% ) = ({\text{C}} - {\text{T}})/{\text{C }} \times 100$$

Where: GI = Growth inhibition (%); C: radial growth of the pathogen in the control; T: radial growth of the pathogen in dual culture (with the antagonist).

After 2–6 days of incubation, the interface region was observed under light microscope. The effect of Trichoderma spp. on microsclerotia production was evaluated by counting the number of microsclerotia. The size of microsclerotia was measured using an ocular micrometer after calibration. The averages of 40 microsclerotia from each treatment were taken to calculate their size.

Fungal growth inhibition by Trichoderma volatile metabolites

Radial growth inhibition of the pathogen through the release of volatile substances was measured, following the method of Dennis and Webster (1971). Briefly, mycelial plug of 5 mm ø of each Trichoderma isolate was placed at the center of plate containing PDA. Then, the lid was replaced by another PDA plate with an M. pseudophaseolina mycelial plug of 5 mm ø at the center. The two plates were sealed together with parafilm and incubated at 25 °C for 5 days, registering mycelium growth every 24 h. As control, a non-inoculated PDA plate was used as a cover. Each test was replicated three times. Radial growth inhibition mycelial was calculated as describe previously in dual culture.

Efficacy of Trichoderma isolates on lentil charcoal rot in vivo

Fungal pathogen inoculum preparation

The pathogenic fungal of M. pseudophaseolina (M1) was used as a target pathogen in this experiment. The microsclerotia suspension was prepared by incubating the pathogenic fungal in potato dextrose agar (PDA) at 28 ± 2 °C for 7 days. The spore suspension was prepared by removing the microsclerotia from the culture edges and adding 5 ml of sterile distilled water for better separation. The suspension was filtered through two layers of sterile cheesecloth, subsequently, was determined to 10⁴ microsclerotia/ml (Khanzada et al. 2012).

Trichoderma inoculum preparation

Trichoderma isolate (T10) was selected as the best and efficient antagonist in this experiment. T10 was grown on PDA plates at 25 °C till the colonies became green. Then, conidial concentration was adjusted to 10⁸ conidia/ml with sterile distilled water (Zaim et al. 2018).

Pot experiment and seed treatment procedure

A pot experiment was conducted in 2019 and 2020 to evaluate the performance of Trichoderma as a biocontrol agent against M. pseudophaseolina. Four treatments and three replicates were done. Lentil seeds (cultivar Syrie 229) were surface sterilized with 1% sodium hypochlorite for 3 min and rinsed three times with sterile distilled water than dried. Each three lentil seeds were sowed in a 7 cm diameter sterilized plastic pot, filled 2/3 full with a sterilized soil mixture and peat (V/V). Sterilization was done at 120 °C for 1 h three times in three days.

The experiment included the following preparations:

Preparation 1 including the non-inoculated control, the seeds were immersed in sterile distilled water without fungal inoculum.

Preparation 2 seeds were immersed only in M. microsclerotia suspension (with pathogen and no Trichoderma).

Preparation 3 including seeds treated with Trichoderma and inoculated with M. microsclerotia suspension (with Trichoderma and pathogen).

Preparation 4 containing separately immersed seeds in conidial suspension of the Trichoderma isolate but without M. microsclerotia of the pathogen (Trichoderma + no pathogen).

Pots were distributed in a completely randomized block design with three replicates per treatment and kept under observation of Charcoal-rot symptoms for 5 weeks compared with the control pots. After 40 days of culture, 5 plants per treatment and control were analyzed. Dry and fresh weight of the aerial part and roots were determined.

Disease assessment

The charcoal rot disease severity (DS) of infection and colonization was assessed as described by Lamini et al. (2020). After 40 days of inoculation, charcoal rot disease symptoms on lentil plants were assessed using disease rating scale from 0 to 5 based on the level of infection and M. microsclerotia presence and abundance observed in stem, root tissues, where 0: no symptoms on plants, 1: minor root infection with few microsclerotia, 2: 30% of root tissue showing lesions, 3: extensive lesions on roots and lower area of stem 4: most of the roots are affected, with high number of microsclerotia on stem and roots, 5: severe infection with plant mortality. Stem colonization results in a considerable amount of microsclerotia produced in the stem. It was measured according to the distance between the collar and the last microsclerotia appearing on the stem.

Disease incidence (DI) was assessed, using the following formula (Lamini et al. 2020):

$${\text{DI}}\% = \frac{{{\text{Number}}\;{\text{of}}\;{\text{infected}}\;{\text{plants}}}}{{{\text{Totale}}\;{\text{number}}\;{\text{of}}\;{\text{plants}}}} \times 100$$

The experiments were conducted using completely randomized block designs.

Molecular identification and phylogenetic analysis of efficient antagonistic Trichoderma T10

For DNA extraction, fungal isolate T10 was cultivated on PDA (slants) at 25 °C for 7 days. The total genomic DNA was extracted from T10 isolate, using the following protocol of the NucleoSpin^® Food commercial kit from Macherey Nagel (Germany). An ITS and TEF regions of the 5.8S rDNA gene were amplified by PCR with universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′), ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and EF1-728F (5′-CATCGAGAAGTTCGAGAAGG-3′) (White et al. 1990; Carbone and Kohn 1999; Elsehemy et al. 2020), in a IcyclerdeBiorad type thermocycler (Thermal Cycler) (Biorad, USA).

A total volume of 25 μl containing 2 μl of T10 genomic DNA, 10 μM of each of the primers indicated above and five units of Taq polymerase (Solis Biodyn) was used to perform the PCR. Conditions for PCR amplification were initial denaturation of 1 cycle at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 45 s; final extension step at 72 °C for 7 min. The amplified products were revealed after electrophoresis on 1.5% agarose gel and purified by Wizard^® SV Gel kit and Promega’s PCR Clean-Up System. The amplifiers b sequenced by Applied Biosystems Big Dye v3.1 kit and PCR primers used for amplification. The sequences obtained were analyzed and cleaned using the CHROMAS PRO software, and then identified by BLAST Program (https://blast.ncbi.nlm.nih.gov/Blast.cgi Blast). Also, a phylogenetic tree was constructed using MEGA X software with Neighbor-joining method based on 1000 bootstrap replicates (Kumar et al. 2018).

Statistical analysis

Collected data from in vitro and in vivo assays were analyzed by statistical variance (ANOVA), using SPSS software (Statistical Package for Social Science) and the treatments were compared by Tukey’s test (p < 0.05). Each treatment consisted of three replicates. The experiment was conducted in a completely randomized design.

Results

Morphological and molecular identification of Trichoderma longibrachiatum

A total of seven Trichoderma isolates were obtained, by dilution isolation technique, from rhizosphere samples of lentil. Colony morphology and morphological features of the isolates typically resembled those of the genus Trichoderma. The T10 was the isolate that had the most important activity antagonist against M. pseudophaseolina, was selected for morphological observations and characterization by ITS and TEF sequences analysis.

T. longibrachiatum produces yellowish green pigmentation at the bottom of the culture plate. The phialids are solitary. The conidia are smooth and ellipsoid (Fig. 1). Chlamydospores are common, terminal or intercalated. The amplification of DNA region with ITS1-ITS4 and TEF1-α primers was successfully used to identify T10. A BLAST search of the obtained sequence revealed 99.83% and 100% similarity with several T. longibrachiatum isolates (MF102214, KY764833, MW193401) and (MF782844, MT881880, MN195113) for ITS and TEF sequences, respectively. A phylogenetic tree built for TEF region sequences with other Trichoderma spp. showed that the T10 isolate was clustered with T. longibrachiatum with a high bootstrap value (Fig. 2). T10 was identified as T. longibrachiatum and the sequence was deposited in NCBI GenBank under accession number MW818102 and OK500004 for ITS and TEF, respectively.

Fig. 1

Morphology of Trichoderma longibrachiatum (T10) a colony on PDA after 4 days, b microscopic observation of the fungus showing phialides and conidia

Fig. 2

Phylogenetic tree of the Trichoderma spp. inferred by Neighbor Joining method of TEF1-α sequence. The bootstrap values from 1000 replication are indicated on the branches

Antagonistic activity of Trichoderma spp. against M. pseudophaseolina

The results of the in vitro antagonistic activity determined by dual culture revealed that from 2 to 6 days after incubation, all isolates of Trichoderma were increasingly effective in inhibiting the mycelial growth of M. pseudophaseolina M1. Table 1 shows the percentage of radial growth inhibition by the different Trichoderma isolates against M. pseudophaseolina M1. All isolates of Trichoderma reduced the mycelial growth of M1 from 37.33 to 72.53%. T10, T5, T6, and T1 isolates were the most effective in vitro, inhibiting M1 growth by more than 50%. The highest inhibitory effect was noted in the isolate T10, in contrast to the isolate T2 showed the lowest inhibitory effect. After 10 days of incubation, the colony of M. pseudophaseolina was totally invaded by T. longibrachiatum (T10) with intense sporulation (Fig. 3c). In addition to mycelial growth, the number and size of microsclerotia were also affected by Trichoderma isolates. The control treatment showed the highest mycelium density and most branched hyphae (Fig. 3a). M1 produced a minimal number of microsclerotia in the presence of T10, followed by T1, causing 68.56 and 53.37% reduction, respectively over the control. On the other hand, T2 caused the minimum reductions of microsclerotia production, followed by T6, resulting in 28.59 and 32.85% reductions, respectively, over the control (Fig. 4a). Moreover, the isolates of Trichoderma spp. can significantly reduce the microsclerotia size of M. pseudophaseolina. The highest reduction in size of microsclerotia was caused by T10, while T6 caused the lowest reduction in size (Fig. 4b).

Table 1 In vitro effect of Trichoderma isolates on Macrophomina pseudophaseolina growth

Fig. 3

Direct in vitro confrontation between Trichoderma longibrachiatum (T10) and Macrophomina pseudophaseolina (M1). a Colony of isolate M1 on PDA after 7 days of incubation. b Direct confrontation between T10 and M1. c Invasion of M. pseudophaseolina colony by T. longibrachiatum after 10 days. d Microscopic observation showing coiling hypha of T. longibrachiatum on M. pseudophaseolina

Fig. 4

Effect of Trichoderma isolate on number and size of microsclerotia of Macrophomina pseudophaseolina

The effect of volatile compounds on M. pseudophaseolina was evaluated to determine the mechanisms used by Trichoderma isolates. As presented in Table 1, all Trichoderma isolates have the capacity to produce volatile substances that inhibit the mycelial growth of M. pseudophaseolina. In comparison to the controls, T1 is the most isolate inhibitor of M1 mycelial growth by volatile compounds, followed by T2, T3 and T10. The inhibition rate varied from 11.11 to 41.66%. T. longibrachiatum (T10) isolate had the greatest antifungal activity against M. pseudophaseolina than the other Trichoderma spp. (Table 1). That is why this isolate was selected for further experiments in vivo.

Efficacy of T. longibrachiatum against M. pseudophaseolina under greenhouse experiment

Biocontrol efficacy of T. longibrachiatum T10 was evaluated in the greenhouse whose seeds were inoculated with M. pseudophaseolina M1 and treated with T10 (Table 2).

Table 2 Effect of Trichoderma longibrachiatum on charcoal rot disease of lentil, and plant growth parameters under greenhouse conditions

Typical symptoms of charcoal rot were noticed in treatments with the pathogen (Fig. 5b). Seedlings of Syria 229 cultivar showing chlorotic foliage, dry stem, and roots. On the other hand, no symptoms were observed on roots and stems when treated with Trichoderma and inoculated with the pathogen (Fig. 5a). The stem colonizing of M1 isolates was recorded at 24%. Figure 5b shows the colonization of lentil stem inoculated with M1 after 21 days. The microsclerotia clearly developed externally to the stem. No disease was noticed in 40 day-old control plants; whereas 91.7% reduction in disease incidence was recorded in the plants inoculated with M1 M. pseudophaseolina and treated T. longibrachiatum T10 as compared to the plants inoculated only with M1.

Fig. 5

Biocontrol efficacy of Trichoderma longibrachiatum species against Macrophomina. pseudophaseolina under greenhouse conditions. a In vivo test, b symptoms of charcoal rot (chlorotic foliage) on lentil plant, c colonization of lentil stem by the pathogen’s microsclerotia

As shown in Table 2, a significant effect of the treatment on the charcoal rot disease incidence (DI) and severity (DS) at 40 days after sowing was recorded. In all seedlings, DI and DS were significantly reduced through the action of seed treated with T10. In fact, the treatment induced a significant decrease in the disease incidence DI values. Meanwhile, the DS values were significantly reduced to 82%. Disease severity score of lentil plants recorded 40 days after the sown has been compiled in (Table 2). Seeds inoculated with M. pseudophaseolina M1 exhibited the highest severity scores (5). Minimum disease severity with 0.9 scores was observed when T10 was applied (significant at p ≤ 0.05).

Growth response measurement of lentil plants

Root alteration of plants lentil as a result of charcoal rot infection affect the root's ability to function properly, resulting in stunted growth and death of seedlings in the severe infection. In light of these changes, it is important to measure the stem and root length, fresh and dry weight of the infected and treated lentil plants in order to determine the degree of damage caused by this disease and the impact of the biocontrol agent on growth parameters. Lentil plant growth was measured 40 days after T. longibrachiatum T10 inoculation. The results pertaining to the efficacy of T10 in disease suppression and plant growth enhancement against the charcoal rot disease of lentil have been compiled in (Table 2). In contrast to untreated plants, the use of T10 with or without M1 significantly (p ≤ 0.05) enhanced the plant growth parameters. Compared to the un-inoculated control, infection by M1 caused significant decreases in plant height (16 cm), root length (14.76 cm), fresh and dry weights (0.1 and 0.04 g/plant, respectively) (Table 2). The seedling growth parameters were significantly increased (p < 0.05) in plants colonized by T. longibrachiatum T10 as compared to untreated seedlings (Table 2). Accordingly, T10 increased the stem and root length by 11.11 and 16.03%, respectively, and fresh and dry weights by 5.88 and 54.54%, respectively.

Discussions

M. pseudophaseolina was first detected on lentil in Algeria in 2021 (Kouadri et al. 2021b). In order to fight against this pathogen, several control methods have been used (Marquez et al. 2021). Therefore, its biocontrol by antagonistic agents such as Trichoderma allows less reliance on chemical pesticides, are commonly employed to control plant pathogens (Topolovec-Pintarić 2019). The use of native isolates in biological control is a crucial factor because they contribute to the balance of agro-systems, while expressing the desired agronomic characteristics such as phytoprotection and phytostimulation of cultivated plants (Zin and Badaluddin 2020). Trichoderma isolates used in the present study are indigenous isolated from the rhizosphere of healthy lentils.

Vinale et al. (2008) have demonstrated that hyperparasitism is connected with the direct contact of an antagonist with a pathogen and is composed of such stages as: pathogen recognition, attack, gradual penetration of the pathogen cells and death.

The present results demonstrated that T. longibrachiatum was the most effective and antagonistic against M. pseudophaseolina. The ability of Trichoderma spp. to overgrow and degrade pathogen mycelia with an inhibition in microsclerotia production was positively correlated with M. pseudophaseolina growth inhibitions during antagonism. The significant antagonistic property of Trichoderma against Macrophomina sp. has been reported by many workers (Bastakoti et al. 2017). Sridharan et al. (2020) reported that the antagonistic activity of T. longibrachiatum caused an inhibition in microsclerotia production of Macrophomina sp.

In dual culture plate tests or through the production of volatile compounds, Trichoderma isolates showed a significant inhibition of Macrophomina sp. growth. Sridharan et al. (2020) demonstrated that antibiosis via microbial volatile organic compounds (mVOCs) was observed in addition to inhibiting the growth of M. phaseolina during a direct interaction with T. longibrachiatum. These mVOCs reduce M. phaseolina mycelial growth by altering the mycelial structure.

Sharma and Sankaran (1988) reported that the fast growth rate of T. longibrachiatum is characteristic of a promising antagonist. According to Sobowale et al. (2010), T. longibrachiatum has a fast and high sporulation rate, which allows it to cover more space per time on a Petri plate; it was also suggested that the competition for nutrients and space is part of inhibition mode of the pathogen.

The activity of Trichoderma isolates in inhibiting M. pseudophaseolina growth is attributed to mycoparasitic activity and production of active compounds, including antibiotics and/or lytic enzymes (Zaim et al. 2018). Many studies have demonstrated that Trichoderma spp. produces a variety of secondary metabolites, some of which are toxic to the pathogens and enzyme production, including chitinases, proteases and glucanases (Gajera and Vakharia 2012). Trichoderma species are also capable of producing cell wall degrading enzymes, such as cellulase, xylanase, pectinase, glucanase, lipase, amylase, arabinase, and protease (Strakowska et al. 2014), as well as many volatile metabolites, such as 6-n-pentyl-2H- -pyran-2-one (6-PAP).

Trichoderma species are also capable of producing a wide range of volatile secondary metabolites such as alcohols, ketones, alkanes, furanes and pyrone 6-PP (6-n-pentyl-2H- -pyran-2-one) which can interfere with various biological processes such as biocontrol and plant growth promotion (Garnica-Vergara et al. 2016). Vinale et al. (2008) reported that 6-PP is one of the major volatile organic compounds VOCs, which has antimicrobial activity at high concentrations and can be involved in plant–fungus signaling leading to the activation of plant defense mechanisms and the regulation of plant growth in tomato, pea, and canola.

Following interaction with Trichoderma isolates, damage on the Macrophomina (M1) microsclerotia was observed. Trichoderma spp. had the ability to reduce the number and size of microsclerotia. Similar results were reported for Trichoderma species having a significant effect on reducing Macrophomina phaseolina microsclerotia size and number (Iqbal and Mukhtar 2020b). Previous published studies gave an evidence that the rhizospheric Trichoderma have been isolated and tested for their antagonistic effects against Macrophomina spp. Several isolates were quite effective in reducing disease incidence and promoting host plant growth traits (Martínez-Salgado et al. 2021).

In most of the earlier studies, Trichoderma mediated plant growth promotion has been attributed to indirect mechanisms viz. control of plant pathogens and induced resistance. The results presented here also demonstrated a significant increase in growth of lentil plants for each of the parameters; plant height, fresh and dry weight, under greenhouse condition.

T. longibrachiatum T10 isolate was selected to be applied in the greenhouse conditions as it recorded the highest inhibition rate accompanied with reduction in size and number of microsclerotia. The obtained results clearly showed that seed treatment with T. longibrachiatum reduced the incidence of charcoal rot disease and improved lentil plant growth parameters. In addition, plants treated with T10 had higher length and weight of stem and root than other treatments. Several reports indicated that Trichoderma species can effectively suppress charcoal rot caused by M. phaseolina on different plants such as soybean and Mung bean (Iqbal and Mukhtar 2020b). Other results obtained by  Khaledi and Taheri (2016) revealed that inoculating soybean seeds with Trichoderma suspension not only suppressed the development of charcoal rot disease, but also increased the activity of peroxidase enzyme and phenolic contents of the soybean roots, resulting in defense activation and plant growth promotion.

Beside the suppression of the pathogen’s growth, the results showed that plant height and weight were increased in plants treated with T. longibrachiatum, and this may be due to the production of secondary metabolites hormones enhancing root and stem growth as well as making some nutrients more available to plants. This is in line with the findings showing that Trichoderma can promote plant growth by producing IAA and ACC deaminase (Ali et al. 2014).

Conclusion

The present study is the first report to use T. longibrachiatum as a biocontrol agent against M. pseudophaseolina, a new pathogen of lentil plants causing charcoal rot disease in Algeria. The results demonstrated that T. longibrachiatum had the ability to control the pathogenic fungus, reduce the infection of charcoal rot on lentil, and promote plant growth. Therefore, these findings strongly support the use of T. longibrachiatum as an effective biological control agent in the management of charcoal rot disease. Future studies will aim to determine the efficacy of the selected isolates under field conditions.