Efficacy of Saccharothrix algeriensis NRRL B-24137 to suppress Fusarium oxysporum f.sp. vasinfectum induced wilt disease in cotton

Anti-Fusarium activity of SA NRRL B-24137 by dual culture plate method

In-vitro, anti-Fusarium activity of SA NRRL B-24137 was evaluated using a dual-culture method containing half of the PDA and half of the ISP2 media. FOV with 10⁶ spore/ml concentration were streaked towards the margin of the Petri dish while the culture of SA NRRL B-24137 was streaked in the center of Petri dish and incubated at 25 °C ± 2 °C for 7 days. The zone of inhibition between the colony margin of SA and FOV was calculated to ascertain the antifungal activity of SA NRRL B-24137 (Hilaire et al., 2015).

Effect of SA NRRL B-24137 on spore germination of FOV

SA NRRL B-24137 antifungal effect on the spore germination of FOV was investigated using the method of Ahmed (2010). FOV spore suspension with concentration of 10⁶/ml (500 µL) was added to SA NRRL B-24137 suspension (500 µL) in glass vials (30 ml) and incubated at 25 °C for 24 h. After 24 hrs incubation, spores in the vials were stained with lactophenol cotton blue and observed for germination status under a microscope. Percentage inhibition was calculated as % Spore inhibition =A − B/A × 100 whereas A: Spore germination in control; and B: Spore germination in treatment.

Anti-Fusarium effect of SA NRRL B-24137 on seed germination

Disease-free, fresh cotton seeds were taken from Ayub Agricultural Research Institute, Faisalabad, Pakistan. Seeds were soaked for 3 min in H₂SO₄ for surface sterilization and rinsed with distilled water in triplicate. After sterilization cotton seeds (n = 07) were placed in a Petri dish containing FOV culture (10⁶ spore/ml) and an equal number of seeds were also placed in a Petri dish containing a mixture of both SA NRRL B-24137 (10⁷ cfu/ml) and FOV (10⁶ spore/ml) culture each with same concentration (500 µL). Both plates were cultured at 25 °C ± 2 °C for 24 h. Then seeds were separated from the culture plates and, after washing, transferred to the sterilized Petri plates containing a double layer of wet blotter paper. After 7 days of incubation, average seed germination was observed in both plates following the method of Jain & Choudhary (2014).

Seed treatment

Sterilized and disease-free, fresh cotton seeds were soaked for 18 h in bacterial SA suspension (10⁷ cfu/ml). In control , seeds were soaked only in carboxymethyl cellulose (CMC) solution. In a sterile setting, a laminar flow hood was used to dry the seeds. Dilution plating suspensions of 10 seeds in 10 ml PB on ISP2 medium yielded a mean bacterial concentration of 1 × 10⁷, which was used to calculate the amount of CFU of bacteria per seed (Erdogan & Benlioglu, 2010).

Biological control of Fusarium wilt in the greenhouse

Seeds treated with bacterial suspension (10⁷ cfu/ml) and control seeds were sown in pots containing autoclave soil (100 g/pot). With the use of a 22-gauge needle, 10 µL conidial suspension of FOV (10⁶ spore/ml) prepared using Hanson’s (2000) technique was injected into the plant body above the soil at the level of the first internode at the six true leaves stage. One drop was also injected into the stele of cotton. At the same time, sterile water is used for the control plants. Pots placed at 27 °C ± 2 °C in the growth room. A scale containing 5 grade (0-4) was used to measure the disease severity both in bacterial treated and control plants (Song et al., 2004).

Disease severity (DS) was determined using equation, ${\displaystyle \text{Disease Severity}=\frac{\left(\Sigma \text{rating no}.\right)\left(\mathrm{no}.\text{plants in rating category}\right)}{\left(\text{Total no}.\text{plants}\right)\left(\text{highest rating value}\right)}\times 10}$

Evaluation of SA NRRL B-24137 biocontrol ability

For six weeks, pot tests were conducted to assess SA NRRL B-24137’s antagonistic effect on FOV. Sterilized seeds were planted in pots (10 seeds/pot, one cm depth) filled with soil that had already been pre-inoculated with FOV (10⁶ conidia/gds), which served as the control group, for this activity. In the experimental group, seeds were sown in pots holding soil pre-inoculated with SA NRRL B-24137 suspension (10⁷ CFU/gds) and FOV (10⁶ conidia/gds). The pots were placed for 3 days in a dark place for maximum germination of seed in the growth chamber.

Evaluation the effect of FOV and SA NRRL B-24137 at different concentrations

The disease severity at various concentrations of FOV (0, 10, 10², 10³, 10⁴, 10⁵–10⁶ conidia/mL) in cotton plants was also scrutinized. Sterilized and healthy seeds were implanted in the earthen pot. When seedlings reached the three true leaves stage, plants were treated with a 25 mL spore suspension of FOV with different spore concentrations (0, 10, 10², 10³, 10⁴, 10⁵–10⁶). All plants were placed in the greenhouse at optimum conditions for 45 days to observe the disease severity and incidence. The experiments were arranged in the following order,

Group 1: Plants without pathogen FOV (control)

Group 2: Plants were infected with 10 spore/ml dose of pathogen FOV

Group 3: Plants inoculated with 10² spore/ml dose of FOV

Group 4: Plants were provided with 10³ spore/ml dose of FOV

Group 5: Plants were given with 10⁴ spore density/ml dose of FOV

Groups 6: Plants were provided with a pathogen dose 10⁵ spore/ml,

Group 7: Plants were provided 10⁶ spore/ml dose of pathogen

To assess anti-Fusarium potential of SA NRRL B-24137, different concentrations of SA NRRL B-24137 suspension (0-10⁷ CFU/mL) were prepared, and seeds were primed with different concentrations for 18 h before sowing in the pot. Plants were infected with various concentrations (10⁴–10⁶) of FOV when these plants reached at three true leaves stage. Disease severity was observed after 45 days of treatment.

Determination of SA NRRL B-24137 and FOV population density in the soil

The population density of SA NRRL B-24137 and FOV in soil was determined up to 9 weeks. SA NRRL B-24137 (10⁷ CFU/gds) was added to the soil, and after 24 h, FOV (10⁶ spore/gds) was also introduced. The serial dilutions of soils on nutrient agar were used to establish the microbial population density in the soil. The colonies of FOV and SA NRRL B-24137 were observed and counted after 3 and 7 days of incubation at 25 °C. Different antibiotics such as 25 mg/L kanamycin, 10 mg/L streptomycin and 50 mg/L chloramphenicol were added to the medium to prevent unwanted microbial growth.

Effect of SA NRRL B-24137 on plant growth

SA NRRL B-24137 effect on different growth parameters of cotton plants was evaluated against FOV. After 45 days of post inoculation, plants were uprooted from the pots and recorded the vegetative growth parameters (plant height (cm), plant weight (gm), root length (cm)). Four treatments were made with three plants in each treatment. Following treatments were used for this experiment.

Treatment 1: Uninoculated control

Treatment 2: FOV

Treatment 3: SA NRRL B-24137 + FOV

Treatment 4: SA NRRL B-24137

After performing all experiments in greenhouse, infected plants and soil were autoclaved before waste to avoid the spread of infection.

Data analysis

Every experiment was carried out in triplicate. The mean ± standard error of the mean is used to express data. GraphPad Prism 5.0 was used to conduct the statistical analysis (Graphpad Prism Software, San Diego, CA, USA). After the analysis of variance, Tukey’s test is conducted. Statistics were judged to be significant for all p-values under 0.05.

Anti-Fusarium activity of SA NRRL B-24137 by dual culture plate method

In-vitro dual culture plate method was used to determine the anti-Fusarium activity of SA NRRL B-24137. The results revealed that SA NRRL B-24137 showed significant antifungal activity against FOV as depicted in Fig. 1. The growth of FOV on a dual culture plate and on control plate (FOV alone) was compared against zone of inhibition.

Effect of SA NRRL B-24137 on spore germination of F. oxysporum

SA NRRL B-24137 antifungal effect on the FOV spore germination was examined. The obtain results showed that SA NRRL B-24137 reduced the spore germination. A positive correlation was also determined with increase the Colony Forming Unit (CFU) of SA NRRL B-24137 the inhibition of FOV spore germination was also increased. SA NRRL B-24137 at 10⁷ CFU/mL gave maximum percentage of FOV spore germination inhibition (87.77%) followed by 10⁶, 10⁵ and 10⁴ CFU/mL as shown in (Fig. 2).

Anti-Fusarium effect of SA NRRL B-24137 on seed germination

Effect of SA NRRL B-24137 on seed germination against FOV was evaluated and results exhibited that FOV have high degree of pathogenicity. An average 25% seed germination was observed in Petri plates containing FOV treated seeds relative to Petri plates containing SA NRRL B-24137 + FOV treated seed with an average of 80% seed germination (Fig. 3).

Exploitation of antifungal potential of SA NRRL B-24137 under greenhouse conditions

After evaluating in-vitro anti-Fusarium potential, the in-vivo efficiency of the SA NRRL B-24137 strain against Fusarium wilt in cotton plants was evaluated under greenhouse conditions. Suspension of SA NRRL B-24137 was prepared and diluted according to requirement, before sowing, cotton seeds were dipped in SA NRRL B-24137 suspension for 18 h. The plants infected with the FOV pathogen showed wilt symptoms after 21 days (Fig. 4A). At the same time, the plants treated with SA NRRL B-24137 before being challenge with the FOV pathogen were healthy in appearance (Fig. 4B).

Disease incidence in the SA NRRL B-24137 treated and challenged with FOV plants was approximately 23%, which was significantly lower than the FOV infected control plants (81%) with rapid wilting of cotton leaves. Specifically, treatment of cotton plants with SA NRRL B-24137 resulted in >74.0% suppression of Fusarium wilt. ANOVA was performed to analyze the results, and a significant difference was observed between the experimental and control groups (Fig. 5).

Evaluation of SA NRRL B-24137 biocontrol ability

The biocontrol potential of SA NRRL B-24137 on disease severity against FOV was recorded up to 6 weeks, and obtained result reveal the remarkable antifungal potential of SA NRRL B-24137 with decreased disease severity gradually in SA NRRL B-24137 treated plants compared to control group (FOV) as shown in (Fig. 6). SA NRRL B-24137 remarkably lowered the soil colonization of pathogen FOV in the pot experiment and subsequently decreased disease incidence over time compared to FOV infected control plants.

After evaluating the effect of FOV and SA NRRL B-24137 on their different concentrations, it was shown that as pathogen FOV dosage (0–10⁶ spore/mL) grew, so did the severity of the disease (Fig. 7A). Moreover, the antagonistic effect of SA NRRL B-24137 was also observed with various concentrations (0–10⁷ CFU/mL) against FOV at 10⁴–10⁷ spore/mL A positive correlation was also found (Fig. 7B). It was confirmed that SA NRRL B-24137 is an effective agent for suppressing Fusarium wilt of cotton caused by FOV but its efficiency differed depending on the concentrations of SA NRRL B-24137 suspension.

Determination of SA NRRL B-24137 and FOV population density in the soil

To assess SA NRRL B-24137 anti-Fusarium activity toward FOV in soil, pot tests were conducted. The initial density of SA NRRL B-24137 (10⁷ CFU/gds) and FOV (10⁶/gds) was introduced into the natural soil. Results showed that SA NRRL B-24137 treatment significantly decreased the FOV soil density and SA NRRL B-24137 remained stable after 9 weeks of 9 weeks” experiment (Fig. 8). In both the sterilized and unsterilized soil, FOV density was calculated, but no discernible change was found (Fig. 9). These findings proved that SA NRRL B-24137 was only responsible for reducing FOV density in the soil and the soil microflora did not interfere in the FOV soil population.

Plant growth promotion (PGP) parameters

During greenhouse experiments, different growth parameters like plant weight, plant length and roots length were also recorded. After 45 days of plantation, plant height, root length and fresh weight of SA NRRL B-24137 treated plants were average 80.67 cm, 50.27 cm, and 14.69 g, respectively while in control plants 44.47 cm plant height, 29.20 cm root length and 8.21 g plant weight, respectively (Fig. 10). Surprisingly, all growth parameters were found higher in only SA NRRL B-24137 treated plants compared to all other groups (Figs. 10 and 11). This might be due to its PGPR activities. Meanwhile, root infected with FOV also differs in morphology as compared to roots morphology of SA NRRL B-24137 treated plants (Fig. 12).