Evaluation of possible biological control of Fusarium concentricum sp. using plant extracts and antagonistic species of microbes in vitro

Collection of Fusarium concentricum

A pure culture of Fusarium sp. was previously isolated and identified (Accession No. MT856371) from postharvest Citrus reticulata fruit rot (Hasan et al., 2020a). The culture was preserved at the Department of Genetic Engineering and Biotechnology, University of Rajshahi (Rajshahi, Bangladesh).

In vitro assessment of antifungal potential of selected plants

For antifungal activities screening, six healthy, mature medicinal plants, Allium sativum, Zingiber officinale, Coccinia grandis, Brassica juncea, Ocimum tenuiflorum and Hibiscus rosa-sinensis were collected from Mirzapur, Binodpur and Kajla village, Motihar, Rajshahi, Bangladesh.

Collected plants were washed with water to remove dust from the plants’ surface and dried in room temperature. Plant extract preparation and fractionation was performed according to the method by Kader et al. (2018). Different parts of selected plants- bulb of Allium sativum; rhizome of Zingiber officinale; leaves of Coccinia grandis, Brassica juncea, Ocimum tenuiflorum; and flowers of Hibiscus rosa-sinensis plants were cut into small species and ground by blender to form fine powder. The dried powder of the plants (100gm of each plant) were rinsed in methanol (500ml) using a conical flask, and were incubated in a shaking incubator with occasional shaking for fourteen days. The liquid contents were pressed through Markin cloth followed by filtration using Whatman no. 1 filter paper. Obtained filtered liquids were dehydrated in vacuo to leave a blackish and sticky mass. The extracts were collected in vials and preserved in a refrigerator at 4°C.

The inhibitory effect of different plant extracts was measured by following the poisoned food technique (Balamurugan, 2014). For this, 20µg of each plant extract was added to 20ml of potato dextrose agar (PDA) to fill a 90mm size Petri plate and mixed well. After solidification, seven day old 6 mm size fungal plug was placed in the center of the Petri plates. The Petri plates were incubated at 35°C for seven days in static condition.

Determination of minimum inhibitory concentration (MIC)

The required MIC values of selected plants extracts were measured through the serial dilution technique (Hussain et al., 2010). The extract of 1 gm of plant-based methanol leaves was dissolved into 1 ml methanol to obtain this same stock solution. Fungal suspension was taken into each test tube and added 50-150µg of plants extracts. The tested tubes were then incubated for 3 days. The progression of the isolated fungus was detected in tubes in which the strength of the substances was just below the inhibition level.

In vitro antagonistic test

For evaluation of antagonistic effects, six non-pathogenic pure microbe cultures, Escherichia coli (pathogenic), Rhizobium phaseoli, Rhizobium leguminosarum, Neofusicoccum mangifera, Trichoderma viride and Pestalotiopsis sp. were used against Fusarium concentricum. The pure microbe cultures were kindly provided by Dr. Md. Salah Uddin, Associate Professor and Director, Microbiology Lab., Department of Genetic Engineering and Biotechnology, University of Rajshahi, as the part of collaboration.

To assess the antagonistic effects, the dual culture technique was used, as previously described (Vethavalli & Sudha, 2012). A mycelial disc of 6 mm diameter was cut from the periphery of both antagonist cultures and the test pathogen and placed on a Petri plate with PDA media. For the control, only the test pathogen was placed in the centre of a Petri plate. The Petri plates were incubated at 35°C in darkness.

Data analysis

The inhibition percentage of mycelial growth= [(Gc-Gt)/Gc] × 100; Where, Gc = Mycelial growth in terms of colony diameter in control set, Gt= Mycelial growth in terms of colony diameter in treatment set. The inhibition percentages of Fusarium species growth were calculated using the following formula: Inhibition percentage (%) =100× (dc– dt)/dc; Where, dc = radial growth of pathogen in control, dt = radial growth of pathogen in dual culture. Mean values were compared through least significant different test using SAS software, version 9.4M5 (SAS Inc., Cary, NC, USA). All the experiment and test were replicated thrice.

In vitro screening of extracts presenting antifungal activity

All plant extracts showed a degree of growth inhibition of the tested fungus at the same concentrations. The highest inhibition of growth of the isolates was observed at 68.1% of mycelium on Coccinia grandis, which was followed by 64.1% on Allium sativum, in comparison to the control culture (100.0%). Hibiscus rosa-sinensis showed the lowest inhibition of mycelium with 29.6% against the fungal isolate in comparison to the control. The results are presented in Figure 1 and Figure 2.

Figure 1. Effect of different methanol plant extracts on percentage inhibition of mycelial growth of Fusarium concentricum.

Mycelia were collected after seven days of incubation on potato dextrose agar at 35°C in darkness. 90 mm Petri plates were used to culture the tested fungus.

Figure 2. Petri dish images showing the effect of plant extracts on inhibition of mycelial growth of Fusarium concentricum.

(A) Allium sativum, (B) Zingiber officinale, (C) Coccinia grandis, (D) Brassica juncea, (E) Ocimum tenuiflorum, and (F) Hibiscus rosa-sinensis. Mycelia were collected after seven days of incubation on potato dextrose agar at 35°C in darkness.

Determination of minimum inhibitory concentration (MIC)

The tested plants extracts showed the MIC values ranges from 80-150µg/ml against the fungus Fusarium concentricum growth. Highest MIC value of isolated fungus was 80µg/ml on Coccinia grandis plant extract and lowest MIC value of isolated fungus was 150 µg/ml on Hibiscus rosa-sinensis plant extract (Figure 3).

Figure 3. Minimum inhibitory concentration (MIC) of methanol extract showing the effect of plant extracts on inhibition of mycelial growth of Fusarium concentricum.

(A) Allium sativum, (B) Zingiber officinale, (C) Coccinia grandis, (D) Brassica juncea, (E) Ocimum tenuiflorum, and (F) Hibiscus rosa-sinensis. Mycelia were collected after seven days of incubation on potato dextrose at 35°C in darkness.

In vitro antagonistic assay

The highest percentage inhibition of radial growth was observed with Trichoderma viride (46.01%) against Fusarium concentricum which was followed by 43.33% and 32.05% on Escherichia coli and Rhizobium phaseoli, respectively (Figure 4). The antagonistic agent Rhizobium leguminosarum did not show any inhibitory activity against the isolated fungus (Figure 4). The control group also did not show any inhibition of radial growth of Fusarium concentricum.

Figure 4. Growth inhibition effects of microbial agents on Fusarium concentricum.

(A) Escherichia coli, (B) Rhizobium phaseoli, (C) Rhizobium leguminosarum, (D) Neofusicoccum mangifera, and (E) Trichoderma viride and (F) Pestalotiopsis sp., Mycelia were collected after seven days of incubation on potato dextrose agar at 35°C in darkness.

Underlying data

Figshare: Effect of plant extracts on inhibition of mycelial growth of the Fusarium species. https://doi.org/10.6084/m9.figshare.13096262 (Hasan, 2020a). This project contains the images of Petri plates for each treatment condition.

Figshare: Effect of antagonistic agents on inhibition of mycelial growth of the Fusarium species. https://doi.org/10.6084/m9.figshare.13096325 (Hasan, 2020b). This project contains the images of Petri plates for each treatment condition.

Figshare: Effects of different plants extract by methanol on inhibition percentages of mycelial growth of the Fusarium species, https://doi.org/10.6084/m9.figshare.13134953 (Hasan, 2020c).

Figshare: Effect of antagonistic agents on inhibition of mycelial growth of the Fusarium species, https://doi.org/10.6084/m9.figshare.13134962 (Hasan, 2020d).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).