Trichoderma longibrachiatum T6 (TL6) was isolated from a rhizisphere soil of a forest site nearby Tianshui, Gansu. Some basic tests were conducted at the Plant Pathology Laboratory of Gansu Agricultural University and it was reported that the strain has no hazardous effects to the environments (Zhuang et al., 2006). The strain of TL6 has since been collected at the China General Microbiological Culture Collection Center, in Beijing, with the patent number (CGMCC No.13183). For the present experiments, the strain of TL6 was cultured on potato dextrose agar (PDA) in Petri dishes for 6 Days at 25°C. The conidia suspension of TL6 was prepared according to the methods described by Zhang et al. (2014b). Final conidia suspension of different densities, 1.5 × 10⁷, 1.5 × 10⁶, 7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ of TL6 conidia per ml, were prepared and stored at 4°C.

Fusarium oxysporum (FO) was obtained from the Plant Pathology Laboratory, Gansu Agricultural University and cultured on potato dextrose agar (PDA) medium at 25°C for 7 Days, and filtered into sterilized beakers. The spore concentration was determined using a hemacytometer, and diluted with sterile water to a final concentration of 1.5 × 10⁷ conidia per ml.

This experiment was replicated three times. The treatments included five different concentrations of TL6 (1.5 × 10⁷, 1.5 × 10⁶, 7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ conidia ml⁻¹) and the two controls with one treated with sterile water and the other treated with F. oxysporum suspension. For each treatment and each replicate, surface sterilized eggs of 100 ± 5 suspended in sterile water were placed in each Petri dish, and counted under a stereomicroscope. After confirmation of the number of eggs in each plate, 5 ml of TL6 suspension were added to the plates for each of the five TL6 concentration treatments, whereas 5 ml of sterile water or F. oxysporum suspension were added to the control plates. Hatching (number of J2s emerged from eggs) and parasitism were recorded on the 8^th Day after the treatment. Percentages of inhibition and parasitism were calculated according to Gao et al. (1998).

Where PPE represents percentages of parasitism on eggs, NEPET number of eggs parasitized in each treatment and TNTE the total number of test eggs.

Where RPIEH represents relative percentages of inhibition of eggs hatching, NJHSWCG number of J2s hatched in sterile water control group and NJHET number of J2s hatched in each treatment.

This experiment was replicated three times. The treatments included five different concentrations of TL6 and the two controls with one treated with sterile water and the other treated with F. oxysporum suspension. For each treatment and each replicate, surface sterilized J2s of 100 ± 5 suspended in sterile water were placed in each 6-well sterilized cell culture plate, and counted under a stereomicroscope. After confirmation of the number of J2s in each plate, 3 ml of TL6 suspension were added to each well for each of the five TL6 concentration treatments, whereas 3 ml of sterile water or F. oxysporum suspension were added to the control plates.

After 24, 48, and 72 h of treatment, the remaining J2s were prodded with a needle and those that did not respond were considered dead. The percentages of immotile nematodes were calculated (Meyer et al., 2004). Linear regression was used to evaluate the effect of different concentrations of TL6 on the corrected motility rate of J2s of H. avenae (Khan et al., 2006). Mortality and the corrected mortality of H. avenae J2s were calculated using the equations described by Zhang et al. (2015).

Where M represents mortality, NDJET number of dead J2s in each treatment and TNTJET the total number of test J2s in each treatment.

Where CM represents corrected mortality, MET the mortality in each treatment and MSWCG the mortality in sterile water control group.

The parasitic effect of different concentrations of TL6 on the J2s of H. avenae was observed every 2 Days after the treatment from the 6^th to 14^th Day, and percentages of parasitism were calculated using the equation of Zhang et al. (2015).

Where PPSSJ represents percentages of parasitism on second stage juveniles, NSSJPET number of second stage juveniles parasitized in each treatment and TNTSSJ the total number of test second stage juveniles.

This experiment was replicated six times with the seven treatments the same as those described above. For each treatment and each replicate, 10 μl (2 ± 1 eggs) of surface sterilized eggs suspended in sterile water were placed in each sterilized Petri dish of 3.5 cm in diameter. After that the suspension of TL6 (990 μl) was added to each sterilized Petri dish of 3.5 cm in diameter for each of the five TL6 concentration treatments, whereas sterile water or F. oxysporum suspension (990 μl) was added to the two separate control plates. The process of infection was observed using a stereomicroscope. The daily observations started on the 2nd Day after the treatment until the 12^th Day. In each day of the observation, the detailed information was recorded for the same eggs.

Following the same protocol of the observation on eggs, we observed the infection of TL6 on J2s for each of the seven treatments and each of the six replicates. Ten μl of suspension containing 2 or 3 J2s were pipetted into each sterilized Petri dish of 3.5 cm in diameter, and TL6 suspension (990 μl) was then added to each culture plate for each of the five TL6 concentration treatments, whereas the same amount of sterile water or F. oxysporum suspension was used in the two controls, respectively. The daily observations started on the 2nd Day after the treatment until the 12^th Day. In each day of the observation, the detailed information was recorded for the same J2s.

Two vital dyes were used to determine the viability of H. avenae eggs and J2s in vitro. Acridine orange (AO, Sigma A6014, US) and neutral red (NR, Solarbio-N8160, Amresco-E89) were dissolved in sterilized distilled water and the dyeing was added to the incubation solution to make (i) 0.01% concentration of AO and (ii) 0.01% concentration of NR. This experiment included two treatments and replicated six times. The first treatment was under ultra-low temperature freezer (−80°C) for 15 min, and the second treatment was with the suspension (1.5 × 10⁷ conidia ml⁻¹) of TL6. The detailed treatment procedures are as follow:

For the first treatment, the suspensions of surface sterilized eggs or J2s (2 ± 1 per 10 μl) in 1.5 ml centrifuge tubes were placed in ultra-low temperature freezer (−80°C) for 15 min, and then moved to a water bath (60°C) for 1 min to thaw quickly. The surface sterilized eggs with the similar stage of growth or J2s were maintained at 4°C as the control. Also, 10 μl of eggs or J2s in each treatment were placed into each well of sterilized cell culture plate (96 well) in the treatment group, and 90 μl of the dye solutions were added to each 96 well cell culture plate, and mixed thoroughly until the dyes fully diffused. The eggs were stained for 15 min whereas J2s were stained for 30 min before being rinsed with sterilized distilled water. The viability of eggs and J2s was identified using an optical microscope.

For the second treatment, the surface sterilized eggs or J2s (2 ± 1 per 10 μl) were placed into each 96 well sterilized cell culture plate, and then the suspension (1.5 × 10⁷ conidia ml⁻¹) of TL6 (90 μl) was added to each plate in the treatment group and sterilized water (90 μl) was added to the control. The eggs and J2s in the treatment and the control group were stained at Day 8 of incubation treatment. The dye solution of 100 μl was added to each 96 well sterilized cell culture plate, and mixed thoroughly until the dyes fully diffused. The eggs were stained for 15 min whereas J2s were stained for 30 min before being rinsed with sterilized distilled water. The viability of eggs and J2s was identified using an optical microscope.

This experiment included seven treatments and replicated three times. The wheat cultivar Yongliang 4, susceptible to H. avenae, was used in the experiment. Wheat seeds were surface sterilized with 1% sodium hypochlorite (NaOCl, 5 min) and sown in 15 cm diameter pots containing 500 g sterile soil (silty clay: sand = 3:1 v/v). Ten seedlings per pot were grown in a greenhouse with air temperature of 25°C ± 0.5, and supplemental day/night lighting of 16/8 h. Pots were irrigated daily with sterilized distilled water which enabled the relative humidity to be maintained around 65%. The experiment was arranged using a completely randomized design. Each replicate had 18 pots, allowing multiple sampling (described below). When seedlings reached 10 cm in height, about 15 Days after sowing, each pot was inoculated with 1,500 ± 100 eggs of H. avenae. Ten Days after the inoculation, seedlings were inoculated with 20 ml of TL6 conidia suspension. The treatments included the five concentrations (1.5 × 10⁷, 1.5 × 10⁶, 7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ conidia ml⁻¹) of TL6 conidia and the two controls (i) seedlings inoculated with eggs but not with TL6 (replaced with sterilized distilled water), and (ii) seedlings not inoculated with eggs or TL6 (Mock-inoculated plants (normal)).

The fresh roots of wheat seedlings were sampled six times at 5 or 10 days intervals (i.e., 5, 10, 15, 20, 30, and 40 Days) after inoculation with eggs and TL6 conidia suspension. Chitinase and β-1, 3-glucanase activity, the contents of lignin and total flavonoids were assayed:

In the study, replicated observations were made randomly and independently of each other and they were in a normal distribution with common variances, thus, the ANOVA assumption was generally met. Our treatments was essentially one factor only, therefore, one-way ANOVA was performed to determine the treatment effect using SPSS Version 16.0 (SPSS Inc., Chicago, IL). The significant differences between the treatments were considered at the level of P < 0.05. Fisher's least significant difference (LSD) values were computed using standard error and T-values of adjusted degrees of freedom. Linear regression was used to determine the relationship between the corrected mortality (%) and the corresponding values of the concentrations of the conidia suspension of TL6. The percentage values were log-transformed prior to statistical analysis.

Overall, the different concentrations of conidia suspension of TL6 (1.5 × 10⁷, 1.5 × 10⁶, 7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ conidia ml⁻¹) had the different inhibitory and parasitic effects on the hatching of H. avenae eggs, with higher concentrations of TL6 presenting a stronger and more significant inhibitory and parasitic effects (Figure 1). At the concentration of 1.5 × 10⁷ conidia ml⁻¹, a large number of conidia of TL6 adhered and surrounded the surface of eggs at the early stages (at Day 2) of infection (Figure 1Ai), and the conidia germinated and produced a large number of hyphae parasitized on the surface at Day 8 (Figure 1Aii). The content of eggs was dissolved by the metabolites of TL6 at Day 12 (Figure 1Aiii). At the concentration of 1.5 × 10⁶ conidia ml⁻¹, many conidia adhered to the surface of eggs at Day 2 (Figure 1Bi). With the increase of incubation time, the conidia parasitized and grew on the surface of eggs. Fewer conidia germinated hyphae and grew on the eggs at Day 8 (Figure 1Bii). The germinated hyphae penetrated into the eggs shell and dissolved the content and eggs shell at Day 12 (Figure 1Biii). As the TL6 concentration was reduced to 7.5 × 10⁵ conidia ml⁻¹, only a few conidia adhered and parasitized on the surface of eggs at Day 2 of inoculation (Figure 1Ci). The hatched nematodes body and the eggs shell were parasitized with the hyphae at Day 8 (Figure 1Cii), and were dissolved at Day 12 (Figure 1Ciii). With the TL6 concentrations were reduced further to 3.0 × 10⁵ and 1.5 × 10⁵ conidia ml⁻¹, only a few conidia surrounded the eggs at Day 2 after inoculation (Figures 1Di,Ei). However, the eggs were parasitized with the conidia and dense mycelium at 3.0 × 10⁵ conidia ml⁻¹ at Day 8 (Figure 1Dii), while the dense mycelium parasitized, penetrated and grew on the eggs at 1.5 × 10⁵ conidia ml⁻¹ at Day 8 (Figure 1Eii). The content and shell of eggs were completely dissolved at the concentration of 3.0 × 10⁵ conidia ml⁻¹ of treatment at Day 12 (Figure 1Diii), whereas the parasitized dense mycelium was grown and penetrated into the eggs shell, and the contents of eggs were dissolved after inoculated with the concentration of 1.5 × 10⁵ conidia ml⁻¹ at Day 12 (Figure 1Eiii). In contrast, the strain of F. oxysporum in the control had a lower inhibitory and parasitic effect on the hatching of H. avenae eggs. Some conidia surrounded the eggs at Days 2 and 8 (Figures 1Fi,ii), but only a few of them geminated hyphae and grew on the surface of eggs at Day 12 (Figure 1Fiii). No inhibitory and parasitic effects were found in the sterile water control. No mycelium was formed and the embryonic development was normal in the sterile water control group (Figures 1Gi,ii) and also nematodes begun to hatch at Day 12 (Figure 1Giii).

With the increase of TL6 concentrations (1.5 × 10⁷, 1.5 × 10⁶, 7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ conidia ml⁻¹), the inhibitory and parasitic effects on the H. avenae eggs hatching rates were increased, while the strain of F. oxysporum in the control had a lower inhibitory and parasitic effect, and the sterile water control had no inhibitory and parasitic effect (Tables 1, 2).

There was a trend that the number of eggs parasitized increased with the increase of TL6 concentrations, and the two highest concentrations of 1.5 × 10⁷ and 1.5 × 10⁶ conidia ml⁻¹ resulted in highest percent parasitism (P < 0.01; Table 1). This effect increased with the increase of the treatment days. In contrast, no fungi parasitized the eggs in the sterile water control. In vitro, with the increase of the TL6 concentrations, the relative percentages of inhibition of eggs hatching were increased (Table 2). The relative percentages of eggs hatching-inhibition were highest at Day 8 (100.0%) and Day 9 (99.2%) and the effect decreased with the treatment days (P < 0.01). Among the five concentrations of TL6, the highest percentages of egg hatching inhibition were obtained with the highest concentrations at 1.5 × 10⁷ conidia ml⁻¹ (P < 0.01).

The different concentrations of conidia suspension of TL6 (1.5 × 10⁷, 1.5 × 10⁶, 7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ conidia ml⁻¹) had the different parasitic and lethal effects on the J2s nematodes (Figure 2). Microscopic examination observed that when the newly hatched J2s were treated with the conidia suspension of TL6, most J2s died, and the conidia adhered at or parasitized on the surface at Day 2 at 1.5 × 10⁷ (Figure 2Ai) and 1.5 × 10⁶ conidia ml⁻¹ (Figure 2Bi), whereas the J2s were treated with lower concentrations (7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ conidia ml⁻¹) became dull, stiff, and showed a wave-like distortion at Day 2, and even a small number of dead J2s were adhered or parasitized by the conidia of TL6 (Figures 2Ci,Di,Ei). At the concentration of 1.5 × 10⁷ conidia ml⁻¹, a large number of conidia of TL6 surrounded or adhered to the surface of J2s at Day 8 (Figure 2Aii), and the surface of J2s was completely parasitized by the conidia of TL6 at Day 12 (Figure 2Aiii). At the concentration of 1.5 × 10⁶ conidia ml⁻¹, a few conidia germinated hyphae and parasitized on the dead nematodes surface at Day 8 (Figure 2Bii), and even the dead nematodes were dissolved by the parasitized hyphae and conidia of TL6 at Day 12 (Figure 2Biii). At the concentration of 7.5 × 10⁵ conidia ml⁻¹, the dead nematodes were parasitized by the conidia and hyphae of TL6 at Day 8 (Figure 2Cii). The body of J2s was completely parasitized by the dense mycelium, and even some conidia started reproduction on the parasitic sites of J2s body. The J2s began to dissolve at Day 12 (Figure 2Ciii). As the TL6 concentration was reduced to 3.0 × 10⁵ conidia ml⁻¹, a few conidia parasitized on the J2s surface, and even germinated hyphae and penetrated into the J2s body at Day 8 (Figure 2Dii), while the parasitized site reproduced a few number of conidia and hyphae which grew on the surface at Day 12 (Figure 2Diii). At the concentration of 1.5 × 10⁵ conidia ml⁻¹, the conidia germinated a small number of hyphae and parasitized on the J2s surface at Day 8 (Figure 2Eii). However, a large number of hyphae penetrated into the integument and the parasitic sites became shrunk by the dense mycelium at Day 12 (Figure 2Eiii). On the contrary, the strain of F. oxysporum had no significant inhibitory and parasitic effect on the J2s of H. avenae, with just a few spores surrounded the J2s at Days 2 and 8 (Figures 2Fi,ii), and only a few geminated hyphae parasitized on the surface of J2s at Day 12 (Figure 2Fiii). The activity, body′s color and shape of J2s in the control group remained intact after inoculated with the sterile water (Figures 2Gi,ii,iii).

With the increase of TL6 concentrations (1.5 × 10⁷, 1.5 × 10⁶, 7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ conidia ml⁻¹), the percentages of parasitism on J2s of H. avenae increased significantly. The percentages of parasitism reached 88.7% after 14 Days of treatment with the concentration of TL6 at 1.5 × 10⁷ conidia ml⁻¹ (P < 0.01), while the strain of F. oxysporum in the control had lower parasitic effect, and the sterile water control had no parasitic effect. Also, with the increase of incubation days, the percentages of parasitism on J2s of H. avenae increased significantly after treated with different concentrations of TL6 (Table 3).

Different concentrations of TL6 showed a significant lethal effect on the activity of J2s, and the highest concentration of 1.5 × 10⁷ conidia ml⁻¹ resulted in highest mortality and corrected mortality (P < 0.01). Moreover, there was a significant linear relationship between TL6 and the J2s corrected mortality, and the increased concentrations of TL6 increased the rates of corrected mortality of J2s. The mortality or corrected mortality increased with the increase of the treatment time when treated with different concentrations of TL6. In contrast, the strain of F. oxysporum in the control had lower lethal effect on the activity of J2s at 1.5 × 10⁷ spores ml⁻¹, and the sterile water control had no significant lethal effect on the J2s mortality regardless of the TL6 concentration (Table 4).

Compared to that of the controls, the color of frozen and parasitized nematodes was significantly different after stained with the dye of AO or NR, respectively. After stained for 15 min, the color of frozen eggs changed to orange (Figures 3Aii,iii,iv) or red (Figures 3Avi,vii,viii), while the color of eggs in control group did not change significantly after stained with AO (Figure 3Ai) or NR (Figure 3Av) for 15 min. Meanwhile, the eggs color changed to orange or red after infected with the conidia suspension of TL6 and stained with AO or NR for 15 min. Especially, the color of stained eggs changed significantly when infected with the conidia (Figures 3Bii,vi) or the mycelium (Figures 3Biii,iv,vii,viii) of TL6, but the color of eggs in control group did not change (Figures 3Bi,v).

The color of frozen J2s changed to orange (Figures 4Aii,iii,iv) or red (Figures 4Avi,vii,viii) after stained with AO or NR, but the color of stained J2s in control group did not change (Figures 4Ai,v). However, the color of stained J2s significantly changed after treated or parasitized with the conidia suspension of TL6. Especially, the color changed quickly and significantly when the conidia (Figures 4Bii,vi) or the mycelium (Figures 4Biii,iv,vii,viii) surrounded and parasitized on the surface of some J2s. In contrast, the color in the control did not change (Figures 4Bi,v).

Sixty-five Days after sowing, an average of 89.7% of wheat seedlings leaves showed etiolation, stunted and turned yellow after inoculated with the eggs of H. avenae, and even averagely 82.6% of leaves wilt readily (Figure 5Ag) compared with mock-inoculated wheat seedlings (Figure 5Aa). However, the wheat seedlings inoculated with eggs of H. avenae grew normally when treated with high concentrations (1.5 × 10⁷, 1.5 × 10⁶, 7.5 × 10⁵, and 3.0 × 10⁵ conidia ml⁻¹) of TL6 (Figures 5Ab–e). For low concentration of treatment (1.5 × 10⁵ conidia ml⁻¹), an average of 23.3% of wheat seedlings leaves showed the symptoms of slight etiolation (Figure 5Af) compared to the control (inoculated with eggs but not with TL6; Figure 5Ag).

Abnormal symptoms of roots included decreased number of roots, highly branched and hyperplasia short lateral roots, and disorder and entanglement of root system after inoculated with eggs of H. avenae (Figure 5Bg). In contrast, these were not observed in mock-inoculated plants (Figure 5Ba). Moreover, the eggs of H. avenae infected roots of wheat seedlings grew normally while treated with the high concentrations (1.5 × 10⁷, 1.5 × 10⁶, and 7.5 × 10⁵ conidia ml⁻¹) of TL6 (Figures 5Bb–d). For low concentrations of treatments (3.0 × 10⁵ and 1.5 × 10⁵ conidia ml⁻¹), an average of 26.5% of wheat seedlings roots appeared slightly decreased in each treatment (Figures 5Be,f), but the symptoms of roots have not appeared highly branched and hyperplasia compared to the control (inoculated with eggs of H. avenae but not with TL6; Figure 5Bg).

Sixty-five Days after sowing, the plant height, root length and fresh weight of wheat seedlings decreased after inoculated with the eggs of H. avenae compared to mock-inoculated plants (P < 0.01), while application of different concentrations of TL6 (1.5 × 10⁷, 1.5 × 10⁶, 7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ conidia ml⁻¹) significantly increased the growth of wheat seedlings, and the effect was more pronounced with the increased concentrations of TL6, compared to the control. The relative growth rates of wheat seedling increased significantly when treated with TL6 at 1.5 × 10⁷ conidia ml⁻¹, and the plant height, root length, shoot and root fresh weight increased by 66.7, 159.3 (Figure 6A), 169.1, and 170.2% (Figure 6B), respectively, compared to the control (P < 0.01). Meanwhile, compared to the mock-inoculated plants, treatments with the concentrations lower than 7.5 × 10⁵ conidia ml⁻¹ had no significant effect on the growth of wheat plants, but had a positive effect on their growth in comparison to the control (Figure 6).

Sixty-five Days after sowing, the number of nematodes in the soil and roots of wheat seedling decreased after the combined application of different concentrations of TL6 and the eggs of H. avenae. Among the five concentrations of TL6, the highest concentration of 1.5 × 10⁷ conidia ml⁻¹ significantly decreased the number of cysts (89.8%) and juveniles (92.7%) of H. avenae in soil (P < 0.01), and the number of juveniles (88.3%), and females (91.3%) in roots after the wheat seedlings inoculated with eggs of H. avenae, compared to the control (P < 0.01) (Table 5).

Compared to the mock-inoculated treatment, the activity of chitinase and β-1, 3-glucanase in wheat seedling roots were increased significantly after inoculation with the eggs of H. avenae (P < 0.01). However, a higher increase in chitinase and β-1, 3-glucanase activity was observed after the wheat seedlings treated with different concentrations of TL6 (1.5 × 10⁷, 1.5 × 10⁶, 7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ conidia ml⁻¹) and eggs of H. avenae. The activity of chitinase and β-1, 3-glucanase reached its maximum at the 20^th and 15^th Days after inoculation with TL6 and thereafter it declined. In the wheat seedlings roots, the maximum activity of chitinase and β-1, 3-glucanase increased from 0.4 to 43.9% (Table 6), and 6.1 to 32.0% (Table 7) for the wheat seedlings inoculated with the different concentrations of TL6 and eggs at the 20^th and 15^th Days, respectively, compared to the control. Specifically, the chitinase and β-1, 3-glucanase activity significantly increased by 43.9 and 32.0% in wheat seedlings roots after the combined inoculation with the eggs and highest concentrations of TL6 at 1.5 × 10⁷ conidia ml⁻¹, compared to the control (P < 0.01).

Total flavonoids in the roots of wheat seedlings increased significantly as results of application of different concentrations of TL6 (1.5 × 10⁷, 1.5 × 10⁶, 7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ conidia ml⁻¹) and inoculation with eggs of H. avenae in comparison to the mock-inoculated plants. Specially, application of higher concentrations of TL6 presented higher total flavonoids in the roots of wheat seedlings, but the lowest concentration increased the total flavonoids contents slightly, compared to the control. The maximum content of total flavonoids in wheat seedling roots was recorded at the 30^th Day after application of different concentrations of TL6 and inoculation with eggs of H. avenae (P < 0.01). Moreover, the total flavonoids in the roots of wheat seedlings increased after inoculation with the eggs of H. avenae alone, compared to that of mock-inoculated plants (Table 8).

The contents of lignin in wheat seedling roots increased after inoculated with the eggs of H. avenae alone, while application of different concentrations of TL6 significantly increased the lignin contents in wheat seedling roots, compared to the mock-inoculated wheat seedlings (P < 0.01). There was a trend that the content of lignin in wheat seedling roots increased consistently with the increase of TL6 concentration or incubation days. The highest increase in the contents of lignin was achieved after application of TL6 at 1.5 × 10⁷ and 1.5 × 10⁶ conidia ml⁻¹, followed by 7.5 × 10⁵, 3.0 × 10⁵, and 1.5 × 10⁵ conidia ml⁻¹, respectively. Compared to the control, the highest increase in the contents of lignin by 34.9% and 32.2% at the 40^th Day after inoculated with eggs and TL6 at the concentrations of 1.5 × 10⁷ and 1.5 × 10⁶ conidia ml⁻¹, respectively (Table 9).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.