Trichoderma based formulations control the wilt disease of chickpea (Cicer arietinum L.) caused by Fusarium oxysporum f. sp. ciceris, better when inoculated as consortia: findings from pot experiments under field conditions

Collection of the pathogen, Fusarium oxysporum and Trichoderma spp.

Chickpea growing fields in District Chakwal, Punjab, Pakistan were surveyed to find Fusarium wilted chickpea plants and collection of soil samples for the isolation of indigenous Trichoderma spp. Three cm of the upper soil was discarded and five sub-samples were taken randomly at a depth of 20 cm and put into sterilized polyethylene bags and were brought to the laboratory for the isolation of indigenous Trichoderma spp. The soil samples from each site were pooled together to give one composite sample for each location (Ru & Di, 2012). Trichoderma spp. were isolated from sampled soil on potato dextrose agar (PDA) by serial dilution technique and the plates were kept at 26 °C for 4 days. The fungal colonies were purified and kept at 26 °C for 7–8 days. The cultures were maintained on PDA slants (Khandelwal et al., 2012). F. oxysporum was isolated from diseased chickpea plant and Trichoderma asperellum was isolated from the rhizosphere of diseased chickpea plant at location 1 (Morat), while Trichoderma harzianum (strain 1) and Trichoderma harzianum (strain 2) were isolated from the soil of location 2 (Dhok Jamal). These locations were ≈ 2.1 km apart from each other.

Media preparation

Glass flasks were washed with tap water and rinsed thrice with distilled water. Peeled potato slices (200 g) were boiled in 500 mL dH2O for 30 min, then filtered through muslin cloth with the extract saved. A total of 20 g dextrose and 20 g agar were added in potato extract and mixed properly to avoid clot formation. Total volume of solution was made up to 1 L by adding distilled water. Media were sterilized for 20 min at 121 °C. Antibiotic streptomycin was added in the media to prevent bacterial growth and the medium was poured in sterilized glass Petri plates.

Isolation of pathogen (Fusarium) from infected chickpea roots

Infected chickpea roots with symptoms of the wilt disease were sectioned (0.5–1.0 cm), washed with tap water, surface sterilized with clorox (NaOCl) for 5 min, rinsed three times with sterilized dH₂O and dried on sterilized filter papers. These root pieces were plated at the rate of five pieces/Petri dish on the PDA medium supplemented with chloramphenicol (0.05 g/L) and incubated at 26 °C for 7 days. The isolated fungi were further sub-cultured on PDA. Pure isolates were observed for their growth patterns and pigmentation on the adverse side of the agar plates. Further microscopic examination was carried out for mycelia and conidial structures using pure culture of F. oxysporum. Pure cultures of the isolated fungi were transferred to PDA slants and stored in a refrigerator at 4 °C. Temporary slide mounts were prepared to confirm identity (Mohamed & Haggag, 2006).

Isolation of Trichoderma spp. from soil through serial dilution method

Stock solution of sample was prepared by dissolving 1 g of soil sample into 10 mL of dH₂O. Serial dilutions of samples were prepared at 10⁻¹, 10⁻², 10⁻³ up to 10⁻⁷. A total of 100 microliters of 10⁻³ of the prepared dilution was spread uniformly on the PDA in a Petri dish with the help of a glass spreader. Plates were covered with parafilm to avoid microbial contamination and placed in an incubator at 26 °C for 3 days. After 3 days, fungal growth was observed on PDA plates (Gil, Pastor & March, 2009).

Purification of fungal spp.

Different fungal cultures were taken from actively growing ends with inoculating needle. Then these fungal cultures were inoculated on to fresh media plates. Petri plates were closed with parafilm and labeled. Plates were incubated in growth chamber and the fungal growth was checked over time.

Identification of Trichoderma spp. and F. oxysporum on morphological basis

Pure cultures of Trichoderma isolates and F. oxysporum were observed for morphological traits e.g., shape and color of colony, spore size, shape and color of conidia and conidiophores, as well as structure and branching of mycelia, based on morphological descriptions (Watanabe, 2010).

Molecular identification of fungal species genomic DNA extraction and PCR of ITS of rDNA

DNA extraction and purification was done by following the procedure as described by (Lacap, Hyde & Liew, 2003; Ghosh & Pal, 2017). The internal transcribed spacer (ITS) regions were amplified by PCR using DNA amplification reagent kit manual (GeNei) with fungal specific forward primer ITS-1 F (Gardes & Bruns, 1993) and the reverse primer ITS-4 (White et al., 1990). PCR was done by adopting the protocol as described by Ghosh & Pal (2017), modified from (Gardes & Bruns, 1993). Amplified products were analyzed on 1% agarose gel containing 12 µL of ethidium bromide in 0.5 X Tris-borate EDTA (TBE) buffer. The purified PCR products were sequenced and obtained sequences were subjected to BLAST (https://www.ncbi.nlm.nih.gov/), and were compared with fungal sequences available at NCBI. The sequences were further manually edited and aligned using CLUSTAL W program in MEGA 11 for phylogenetic analysis and to study the diversity among tested isolates.

Dual culture experiments

The Trichoderma spp. were initially assessed for their antagonistic activity against F. oxysporum by in vitro dual culture technique. A 5 mm diameter mycelial disc from the margins of the 7 days old culture of Trichoderma spp. and the F. oxysporum were placed on the opposite of the plate at equal distance. The Petri plates for each test isolate were arranged in a completely randomized design (CRD) with three replicas and incubated at 25 ± 1 °C for 7 days. Radial growth was measured after 7 days of the incubation period and % age inhibition was determined as follows;

L = [(C − T)/C] × 100

where L = inhibition of mycelial growth; C = growth measurement of the pathogen in control and T = growth of the pathogen in the presence of Trichoderma (Wonglom et al., 2019).

Mass culturing of Fusarium oxysporum and Trichoderma spp.

For mass culturing of F. oxysporum, sorghum seeds were autoclaved in heat resistant polythene bags and were inoculated with spores of F. oxysporum, under aseptic conditions, in a laminar air flow cabinet. These bags were incubated at 26 ± 2 °C for ten days until all the grains were fully covered with fungal spores/mycelia. After 10 days, grains were air dried and ground to powder.

Preparation of carrier based Trichoderma formulations and Fusarium inocula

Mass production of Trichoderma spp. included talc based formulation. In talc based formulation, PDA was used for initial growth of Trichoderma spp. in Petri plates. A total of 1,000 mL PDA was prepared in conical flask. Flask was plugged with cotton and covered with aluminum foil to avoid air-borne contamination & sterilized by autoclave at 15 lbs (121 °C temperature) for 15 min. PDA medium was poured in Petri dishes and allowed to solidify. Trichoderma spores were transferred in Petri plates and incubated at 25 ± 2 °C for 6 days. When Trichoderma covered the whole plate, then spore suspension was prepared. One mL of dH₂O was added in Trichoderma culture with the help of a pipette. Trichoderma spores were suspended in 100 mL distilled water. After washing of chickpea seeds, these seeds were soaked in 2% sucrose solution for 6 hrs (Batta, 2004). Then extra sucrose solution was removed from chickpea seeds. These seeds were packed within polypropylene bags and sealed. PP-bags were autoclaved at 15 lbs (121 °C) for 15 min. After sterilization, one mL spore suspension was added on autoclaved chickpea seeds and incubated at 25 ± 2 °C for 15 days. After fifteen days, Trichoderma impregnated chickpea seeds were ground in a mixer. Fusarium inoculum was prepared in the same manner as described for Trichoderma, except that Fusarium mass multiplication was made on sorghum seeds (Shanmugam, Chugh & Sharma, 2015).

A total of 2 gm culture of each isolate was mixed well with 100 mL dH₂O and filtered through muslin cloth. Few drops of Tween 20 (Alkest TW 20) were added in the spore suspension as a wetting agent (Jamil et al., 2010). Spore concentration was measured/gram of chickpeas using hemocytometer. Then the required quantities of chickpeas with Trichoderma inoculum were added into 1 kg sterilized talc [Mg₃Si₄O₁₀(OH)₂] (Batch # S.15442; Rurka Export China Food and Pharma Grade Osmanthus Brand), and 5 gm of carboxymethyl cellulose was applied to seeds and mixed thoroughly to get fine coating on seeds. The treated seeds were spread over blotter paper and air dried (Pandey, Gohel & Jaisani, 2017). The product was packed in PP-bags and sealed. The final colony forming units (CFU) in all Trichoderma spp. were adjusted at 1.5 × 10⁷ CFU/g of product, while, the final CFU in F. oxysporum was adjusted at 1.2 × 10⁵ CFU/g of the product. Fine coated seeds were sown in each sterilized pot (Bhagat & Pan, 2011).

Pot experiments

Pot experiments were performed to check the efficacy of Trichoderma as biocontrol agent against Fusarium wilt. Chickpea susceptible variety (Pb-91) (Rashid et al., 2013) was chosen as a test crop. Certified seeds of chickpea variety were purchased from the local market.

Preparation and sterilization of soil mixture

Silty loam soil collected from a field at National Agricultural Research Centre (NARC) Islamabad, Pakistan was passed through a sieve. The soil and river sand were mixed in a ratio of 4:1 and filled in sacks and autoclaved. Sterilized earthen pots were filled with 1.5 kg of soil (Siddiqui & Singh, 2004).

Raising and maintenance of chickpea plants in pots

Chickpea seeds were disinfected with 0.1% mercuric chloride and rinsed thrice with autoclaved water. Five seeds were sown (during the month of November each year) into each pot at equal distance from each other. Thinning was carried out after successful emergence of seedlings to three chickpea plants per pot.

Following treatments were investigated in pot experiments;

T1 = (Non-infested control) without Trichoderma and Fusarium

T2 = (Infested control) Fusarium (1.2 × 10⁵ CFU/g of product), 10 g

T3 = Fusarium (1.2 ×10⁵ CFU/g of product), 10 g + Trichoderma asperellum (1.5 × 10⁷ CFU/g of product) 8 g (concentration 1)

T4 = Fusarium (1.2 × 10⁵ CFU/g of product), 10 g + Trichoderma asperellum (1.5 × 10⁷ CFU/g of product) 12 g (concentration 2)

T5 = Fusarium (1.2 ×10⁵ CFU/g of product), 10 g + Trichoderma harzianum (strain 1) (1.5 × 10⁷ CFU/g of product) 8 g (concentration 1)

T6 = Fusarium (1.2 × 10⁵ CFU/g of product), 10 g + T. harzianum (strain 1) (1.5 × 10⁷ CFU/g of product) 12 g (concentration 2)

T7 = Fusarium (1.2 × 10⁵ CFU/g of product), 10 g + Trichoderma harzianum (strain 2) (1.5 × 10⁷ CFU/g of product) 8 g (concentration 1)

T8 = Fusarium (1.2 × 10⁵ CFU/g of product), 10 g + T. harzianum (strain 2) (1.5 × 10⁷ CFU/g of product) 12 g (concentration 2)

T9 = Fusarium (1.2 × 10⁵ CFU/g of product), 10 g + T. asperellum, T. harzianum (strain 1), T. harzianum (strain 2) consortium (1.5 × 10⁷ CFU/g of product) 2.66 g of product of each Trichoderma (concentration 1)

T10 = Fusarium (1.2 × 10⁵ CFU/g of product), 10 g + T. asperellum, T. harzianum (strain 1), T. harzianum (strain 2) consortium (1.5 × 10⁷ CFU/g of product) 4 g of product of each Trichoderma (concentration 2)

Experimental design was CRD. Pot experiments were executed in two consecutive seasons (2020–2021 & 2021–2022). Each treatment was replicated three times. Field related experiments were approved by Crop Diseases Research Institute (CDRI), National Agricultural Research Centre, Islamabad, vide Dy No. 10712.

Disease measurements, disease incidence and severity

In case of disease measurements in pot experiments, disease incidence (DI) and disease severity (DS) were determined (Khan, Khan & Mohiddin, 2004; Nandeesha & Huilgol, 2021).

Following formula was used for determination of DI and DS. For DS, 0–5 scale was adopted; 0 = no wilt, 1 = 1–20%, 2 = 21–40%, 3 = 41–60%, 4 = 61–80%, & 5 = 81–100% (Khan, Khan & Mohiddin, 2004; Jamil & Ashraf, 2020). These data in pots were recorded 68 days after sowing (DAS), in the years 1 & 2.

Wilt incidence (%) = $\frac{\text{Total}\text{number}\text{of}\text{wilted}\text{plants}}{\text{Total}\text{number}\text{of}\text{plants}\text{observed}}\times 100$

Disease severity = Number of branches, twigs, or leaves showing wilt symptoms/total number of branches, twigs, or leaves.

Harvesting and data collection in pot experiments

Pot data were taken after 101 DAS, during the month of February each year. Following morphological parameters were recorded in pot experiments; shoot length, shoot fresh and dry weights. In case of disease measurements, DI and DS were determined (Dubey, Suresh & Singh, 2007).

Soil analyses

Soil analyses of chickpea growing fields used to isolate Fusarium and indigenous Trichoderma spp. and of pot soils were carried out by following the procedures as described by Estefan, Sommer & Ryan (2013) (Table 1).

Environmental data

Environmental data were collected from the Pakistan Meteorological Department, Islamabad, Pakistan (Table 2).

Confirmation of Koch’s postulates

To fulfill Koch’s postulates, healthy chickpea plants were inoculated with F. oxysporum grown on a culture that had been isolated from Fusarium wilt affected chickpea root. Then the plant was studied for disease symptoms and its comparison to the infected plants that had been used for the isolation of F. oxysporum.

Statistical analyses

All the data were analyzed by ANOVA, and after performing ANOVA, Fisher’s LSD test was performed at P = 5%, using Minitab 20.2.

Morphological identification of isolated Fusarium and Trichoderma strains

F. oxysporum showed two types of conidia; macroconidia were boat shaped and four celled, while microconidia were ellipsoidal shaped and one celled. Chlamydospores were brown in color and globose shaped. Colony was white in color. Microconidia were with size of 6.1 µm × 3.2 µm. While, macroconidia were sickle shaped with an average size of 30.7 µm × 3.4 µm.

T. harzianum showed conidiophores and hyaline phialides were short and thick. Conidia were ovate shaped and one-celled. Conidiophore size was 78 µm, while conidia were 2.4 µm in diameter. Colony was dark green in color on growth medium. On the other hand, T. asperellum had slightly ovoidal shaped conidia, having size of 2.91 µm × 2.37 µm. Conidia were one-celled.

Phylogenetic analysis of Trichoderma spp.

Phylogenetic analysis had shown that the three isolates of the Pakistani Trichoderma collected for this study clustered in three clades with reference sequences from different regions of the world. The Trichoderma sp. (SA623043) reported in this study clustered with a sequence of Trichoderma from Pakistan, China, and Africa with 99% bootstrap value. The Trichoderma sp. (SA1522759) reported in this study clustered with two sequences of Trichoderma sp. from Nigeria and Brazil that was part of another clade with sequences from Brazil and Nigeria. The third isolate of Trichoderma sp. (W116499) reported in this study clustered with number of sequences of Trichoderma from Pakistan, China, Portugal, and Taiwan with 99% bootstrap value. The phylogenetic framework based on ITS sequences clarified the T. harzianum, although belonging to the same species, yet they phylogenetically belong to different clades (Fig. 1).

Phylogenetic analysis of F. oxysporum f. sp. ciceris

Phylogenetic analysis had shown that the isolate of the Pakistani F. oxysporum f. sp. ciceris analysed in the present study grouped in the major clade with reference sequences from Pakistan, India, Egypt, Turkey, and China with 48% bootstrap value. The F. oxysporum f. sp. ciceris (OR808009) reported in this study clustered in a sub-clade with a sequence of Fusarium from India and Egypt.

The evolutionary history was deduced with Neighbor-Joining technique (Saitou & Nei, 1987). The % age of replica trees in which the associated taxa clustered together in the bootstrap test (500 replicas) are shown next to the branches (Felsenstein, 1985). The evolutionary distances were computed by the Maximum Composite Likelihood technique (Tamura, Nei & Kumar, 2004) and are in the units of number of base substitutions per site. This analysis involved 17 nucleotide sequences including nucleotide sequence (OR808009) reported in present study. All obscure positions were removed for each sequence pair (pairwise deletion option). There were a total of 1,244 positions in the final dataset. Evolutionary analyses were performed on MEGA 11 (Tamura, Stecher & Kumar, 2021) (Fig. 2).

Effect of various Trichoderma spp. on Fusarium in dual culture experiments

In dual culture experiments, T. asperellum inhibited the F. oxysporum f. sp. ciceris by 37.6%, while, T. harzianum strains 1 and 2 significantly inhibited the radial/mycelial growth of F. oxysporum by 40% and 42%, respectively (Table 3).

Effects on shoot length, shoot fresh weight, and shoot dry weight of chickpea plant in pot trials (year 1)

The Fusarium infested chickpea plants (infested control, IC) showed a 27% decrease in shoot length as compared to non-infested control (NIC). Treatments with T. asperellum (T.a) concentration 1 and concentration 2, T. harzianum strain 1 (Th S1), concentration 1 and concentration 2, T. harzianum strain 2 (Th S2) concentration 1 and concentration 2, and consortium of all these Trichoderma species (concentration 1&2), significantly increased the shoot length of chickpea plants by 20% and 31%, 32% and 39%, 39% and 46%, 56% and 69%, respectively, over IC. Moreover, consortium of all these Trichoderma species at concentrations 1 and 2, significantly enhanced the shoot length of chickpea plants by 13% and 23%, respectively, in comparison with NIC (Fig. 3A).

The chickpea plants that were infested with Fusarium experienced a 43% decrease in the shoot fresh weight as compared to NIC. However, all treatments involving Trichoderma showed a significant increase in shoot fresh weight, in comparison with IC, effectively counteracting the negative effects of the F. oxysporum pathogen. The effectiveness of Trichoderma in controlling F. oxysporum was found to depend on the dosage. Treatments with T.a, Th S1, Th S2, and a combination of these Trichoderma species, all significantly increased the shoot fresh weight of chickpea plants. These increases were 21% and 32%, 31% and 37%, 38% and 44%, and 60% and 67% at concentrations 1 and 2, respectively, compared to the IC (Fig. 3B).

IC of chickpea plants, affected by Fusarium, exhibited a 41% decrease in shoot dry weight, when compared to the NIC. However, all treatments involving Trichoderma showed a significant increase in shoot dry weight, effectively mitigating the negative effects of the pathogen, F. oxysporum. The efficacy of Trichoderma in controlling F. oxysporum was found to be dependent on the dosage. Treatments with T.a, Th S1, Th S2, and a consortium of these Trichoderma species significantly increased the shoot dry weight of chickpea plants. The increases were 22% and 32%, 32% and 38%, 37% and 42%, and 56% and 68% at concentrations 1 and 2, respectively, compared to the IC. Furthermore, the consortium of all these Trichoderma species at concentrations 1 and 2 showed non-significant differences when compared to the NIC (Fig. 3C).

Effect on disease incidence and disease severity of chickpea plants in pot trials (year 1)

There was 88.9% disease incidence (DI) in Fusarium infested chickpea plants, as compared to NIC. All the treatments with Trichoderma demonstrated a significant decrease in DI of chickpea plants. Treatments with T.a, Th S1, Th S2, and consortium of all these Trichoderma spp. significantly decreased the DI of chickpea plants by 77.8% and 66.7%, 66.7%, 55.6% and 44.4%, 33.3%, 33.3% and 22.2%, at concentrations 1 and 2, respectively, over IC. Moreover, consortium of all these Trichoderma spp. at concentrations 1 and 2, significantly reduced the DI on chickpea plants by 33.3% and 22.2%, respectively, in comparison with infested control (IC).

In year 1, IC had a disease severity (DS) of 4.7, whereas, there were no disease symptoms in NIC. Moreover, all the treatments using Trichoderma showed significant reduction on DS in the chickpea plants and this reduction in DS was dose dependent. Treatments using T.a, Th S1, Th S2, and a combination of all these Trichoderma, all significantly decreased the DS in the chickpea plants. The reductions in DS were 21% and 23%, 29%, 31%, and 40%, and 47%, 71%, and 86% at concentration 1 and concentration 2, respectively, compared to the IC (Table 4).

Effect on disease incidence and disease severity of chickpea plants in pot trials (year 2)

There was 77.8% DI in Fusarium infested chickpea plants (IC), as compared to NIC. All the treatments with Trichoderma demonstrated a significant decrease in DI of chickpea plants. The increase in the concentration of Trichoderma resulted in a decrease of DI. Treatments with T.a, Th S1, Th S2, and consortium of all these Trichoderma species significantly decreased the DI of chickpea plants by 66.7% and 55.6%, 55.6%, 44.4% and 44.4%, 33.3%, 22.2% and 11.1%, at concentrations 1 and 2, respectively, over IC. Moreover, consortium of all these Trichoderma spp. at concentrations 1 and 2, significantly reduced the DI of chickpea plants by 22.2% and 11.1%, respectively, in comparison with infested control (IC).

In year 2, IC revealed DS score of 4.1. Trichoderma showed a significant decrease in DS caused by the pathogen in the chickpea plants. Treatments using T.a, Th S1, Th S2, and a combination of all these Trichoderma species significantly reduced the DS in the chickpea plants. The reductions in DS were 14% and 30%, 30%, 35%, and 35% at concentration 1, and 57%, 68%, and 92%, at concentrations 1 and 2, respectively, in comparison with IC (Table 4).

Effects on shoot length, shoot fresh weight, and shoot dry weight of chickpea plants in pot trials (year 2)

In year 2 pot experiments, infested control showed a 23% decrease in shoot length as compared to NIC. All the treatments with Trichoderma demonstrated a significant increase in shoot length of chickpea plants, minimizing the negative effects of the pathogen, F. oxysporum. The effect of Trichoderma in controlling the F. oxysporum was found dose dependent. Treatments with T.a, Th S1, Th S2, and consortium of all these Trichoderma species significantly increased the shoot length of chickpea plants by 26% and 36%, 44% and 47%, 48% and 54%, 64% and 72%, at concentrations 1 and 2, respectively, over IC. Moreover, consortium of all these Trichoderma species at concentrations 1 and 2, significantly enhanced the shoot length of chickpea plants by 27% and 33%, respectively, in comparison with NIC (Fig. 4A).

The Fusarium infested chickpea plants revealed 38% decline in the shoot fresh weight compared to NIC. All treatments involving Trichoderma either alone or in the form of consortia, showed significant enhancement in shoot fresh weight of chickpea plants, indicting the beneficial effect of Trichoderma against F. oxysporum. Treatments with T.a, Th S1, Th S2, and a combination of these Trichoderma species, all significantly increased the shoot fresh weight of chickpea plants. The increases were 28% and 37%, 45% and 50%, 50% and 57%, and 67% and 73% at concentrations 1 and 2, respectively, compared to the IC. Furthermore, the combination of all these Trichoderma species at concentrations 1, 2 and Th S2 showed non-significant differences, in comparison with NIC (Fig. 4B).

Fusarium infection exhibited a 36% decrease in shoot dry weight of chickpea plants as compared to NIC. Treatments with T.a, Th S1, Th S2, and a consortium of these Trichoderma species significantly increased the shoot dry weight of chickpea plants by 25% and 36%, 44% and 49%, 56% and 63%, and 68% and 75% at concentrations 1 and 2, respectively, compared to the IC (Fig. 4C).

Soil analyses

Soil texture was sandy loam at chickpea growing locations as well as the soil used in pot experiments. There was significant difference in the soil pH and soil organic matter (SOM) of both locations, while the pH and SOM of soil used in pot experiments in both years were non-significant, when compared with each other. NO₃-nitrogen, available phosphorus, available potassium, iron, and copper were significantly different at both locations, except zinc. On the other hand, NO₃-nitrogen, available phosphorus, iron, and copper were significantly different in the soils used in pots of year 1 and year 2, except available potassium and zinc. Moreover, variable results were recorded when we compare the soil properties used in pot experiments as compared with soil properties of location 1 and 2 (Table 1).

Environmental data

Environmental data for the years 2020–2021 and 2021–2022 showed that during the critical stage (flowering stage susceptible to Fusarium wilt = January), there was high rainfall (Table 2).

Confirmation of Koch’s postulates

The healthy chickpea plants inoculated with F. oxysporum grown on a culture that had been isolated from Fusarium wilt affected chickpea root in the pot trials, developed the same symptoms as inoculated initially.