Index

Abstract

An in-vitro experiment was conducted at National Centre for Genetic Resources and Biotechnology and the Nigeria Agricultural Quarantine Service, Moor plantation, Ibadan. The experiment was carried out to test the antifungal efficacy of some plant extracts (Ageratum conyzoides, Azadirachta indica, Morinda lucida, and Chromolaena odorata) and a chemical fungicide (mancozeb). The mycelial growth inhibition potentials of five concentrations of aqueous plant extracts were assayed at different incubation periods on the growth of Colletotrichum orbiculare and Lasiodiplodia theobromae. The experiment was carried out in a Completely Randomized Design (CRD) with five replications. A 3 mm mycelial disc of each test fungus was placed at the center of a 9 cm Petri dish containing 5, 10, 15, 20, and 25 g of the plant extracts or 0.25g/100mL of mancozeb (synthetic fungicide) in Potato Dextrose Agar. The results obtained revealed that all the plant extracts, at all concentrations, significantly inhibited the growth of these mycopathogens, with 25g C. odorata having the highest percentage inhibition of 70.78% and 73.68% at 48 and 96 hours of incubation on C. orbiculare and L. theobromae respectively. All the selected aqueous extracts inhibited more than 50% C. orbiculare mycelial growth. Antifungal extracts recovered from the selected plants could be further purified to improve and characterize their fungicidal activities in controlling plant diseases. Extracts of plant materials, which are readily available to the farmers, are better alternatives to the commonly used hazardous, synthetic fungicides.

Keywords: Plant extracts, Inhibition, Lasiodiplodia theobromae, Colletotrichum orbiculare, Mycelial growth, In-vitro.

Received: 8 January 2021 / Revised: 5 February 2021 / Accepted: 26 February 2021/ Published: 11 March 2021

Contribution/ Originality

This paper’s primary contribution is finding that all the plant extracts used against cucumber pathogens had significant inhibitory properties inhibited at different concentrations and incubation periods respectively.


1. INTRODUCTION

Cucumber in Nigeria is fast becoming one of the important vegetables produced, being a source of proteins, carbohydrates, minerals, vitamins, and fibers (Abbey, Nwachoko, & Ikiroma, 2017). Despite its importance, fungal diseases are one of the factors that affect its production.  Many pathogens are known to attack cucumber but Colletotrichum orbiculare and Lasiodiplodia theobromae are among the most destructive pathogens that attack this vegetable crop. In recent times, a lot of investigations were done on the use of extracts of medicinal plants for controlling many phytopathogens (Bobbarala, Katikala, Naidu, & Penumajji, 2009). Most plant constituents such as alkaloids, glycosides, terpenes, terpenoids, and different flavonoids have been reported to show high potential effects against plant diseases which were extracted from plants such as Laurus camphora L., Peganum harmala L., Zygophyllum coccineum L. and Cymbopogon nardus C. (Abeer, Hoballah, Abdel-Halim, & Sanaa, 2017). Chemical fungicides have been employed over the years for the control of fungal diseases to increase crop production. Unfortunately, its overzealous and indiscriminate use has created different environmental and toxicological problems and the occurrence of resistant strains of pests (Gurjar, Ali, Akhtar, & Singh, 2012). This led to the development and utilization of newer approaches, including the use of botanical pesticides which are eco-friendly. Pesticides obtained from plants are effective in the management of plant pathogens and biodegradable, thereby supporting both crop production and the environment than synthetic fungicides (Utobo, Ekwu, Nwogbaga, & Nwanchor, 2016). Phytochemicals present in these botanical pesticides have proved to have inhibitory effects on all types of phytopathogens (Gurjar et al., 2012). Therefore, this present study was designed to investigate the efficacy of aqueous extract of different plant materials at various concentrations and incubation periods on the in-vitro control of Colletotrichum orbiculare and Lasiodiplodia theobromae.

2. MATERIAL AND METHODS

2.1. Sample Collection and Isolation of Fungal Isolates

The plants used in this study (Ageratum conyzoides, Azadirachta indica, Morinda lucida, and Chromolaena odorata) were obtained from the Federal College of Agriculture, Ibadan. Cucumber leaves showing various disease symptoms of anthracnose were obtained from the cucumber field at Abeere, Ede North local government, and Isale Osun, Osogbo local government area of Osun State, Nigeria. The infected leaves were collected in sterile bags and taken to the laboratory for isolation and identification of pathogens. Infected leaves were dissected with a sterile scalpel at the interphase between the healthy and necrotic portions of the leaves, surface sterilized with 2 mL sodium hypochlorite solution for 2 min, and rinsed in four successive changes of sterile distilled water (SDW) (Gwa & Nwankiti, 2017). Disinfected tissues were blotted with sterile filter paper for 2-3 min in the laminar airflow cabinet to dry, aseptically plated on Petri dishes containing acidified sterile potato dextrose agar (PDA), and incubated at room temperature for 7 days.

2.2. Characterization and Identification of Fungal Isolates

Pure cultures were obtained following the sub-culturing of the growing fungi after 7 days of incubation. Microscopic examination and morphological characteristics were noted and compared with existing authorities (Burgess, Knight, Tesoriero, & Phan, 2008).

2.3. Pathogenicity Test

The experiment was carried out according to Dania, Fadina, Ayodele, and Kumar (2015) using, completely randomized design, replicated five times. Planting pots with the dimension 21.5 cm x 27 cm were filled with 7 kg of sterilized soil; three seeds of each variety of cucumber were sown in each pot and later thinned to 2 seedlings per pot. After three weeks of planting, 6.0 x 106 spore/mL suspension of the fungal isolates was sprayed on cucumber seedlings. Six days after inoculation, plants that showed symptoms of infection were collected and taken to the laboratory for further evaluation.

2.4. Preparation of Plant Extracts

The plant extracts were prepared according to the methods described by Tohamy, Aly, Abd-El-Moity, Atia, and Abed-Rl-Moneim (2002). Azadirachta indica, Ageratum conyzoides, Morinda lucida, and Chromolaena odorata leaves were washed thoroughly with cold running tap water, air-dried to constant weight at room temperature, and separately grounded into a fine powder using a Warring blender. Different weights of each plant powder (5 g, 10 g, 15 g, 20 g, and 25 g) were soaked in 100 mL of sterilized distilled water for 24 hours and subsequently filtered through  a four-fold of sterile cheese-cloth and 1.25 mm Whatman filter paper. The filtrates were used as the plant extracts in the experiment. Also, 0.25 g of Mancozeb (fungicide) was added to 100 mL of distilled water. Thereafter, 1 mL of each concentration of the extracts or chemical fungicide was added to sterilize PDA.

2.5. Effect of Plant Extracts on Mycelial Growth of Fungal Isolates

To evaluate the fungi toxic effect of the plant extracts and the chemical fungicide on fungal mycelial growth, cool (about 45ºC), molten PDA medium with the plant extracts were mixed and allowed to solidify before the inoculation of fungi (Gwa & Nwankiti, 2017). The experiment was carried out using a completely randomized design with 5 replications. To create a point of intersection that would indicate the center of the plates, two perpendicular lines were drawn at the bottom of the Petri dishes to generate four equal sections on each plate (Amadioha & Obi, 1999). Three milliliters of each plant extract and chemical fungicide, at different levels of concentrations, were poured into Petri dishes containing 9 mL of the sterilized PDA, properly swirled, and allowed to solidify (Nene & Thapilyal, 2002). Five - millimeter mycelial disc of the test fungi (pure cultures) were thereafter inoculated at the point of intersection drawn at the bottom of the plate. Sterile PDA without plant extracts and chemical fungicide served as the control treatment. The inoculations were incubated at room temperature (25±2ºC) and the growth was monitored. The mycelial radial growth of the fungal isolates was recorded at 48, 72, 96, 120, and 144 hours after inoculation.  

Fungal inhibition was determined as percentage growth inhibition (PGI) according to the method described by Iwuagwu, Onejeme, Ononuju, Umechuruba, and Nwogbaga (2018).

Where,
PGI = Percent Growth Inhibition.
R = the distance (measured in mm) from the point of inoculation to the colony margin in the control plate.
R1 = the distance of fungal growth from the point of inoculation to the colony margin in the treated plate.

2.6. Data Analysis

Data collected were analyzed using the analysis of variance (ANOVA) at P ≤ 0.05 and mean separation was done using Duncan multiple range test.

3. RESULTS

Results of the inhibitory effect of different concentrations of selected plant extracts on cucumber pathogens revealed that mancozeb, the synthetic chemical used in this study, completely inhibited the growth of the pathogens. All the plant extracts (C. odorata, M. lucida, A. conyzoides, and A. indica) significantly exhibited antifungal properties against the fungi. The results revealed that the higher the concentration of the plant extracts, the higher the rate of inhibition of the mycelia growth of these pathogens. Table 1 shows the results of different plant extracts on mycelia growth of C. orbiculare at different concentrations. At 96 hours of incubation, the result revealed that 25 g of C. odorata had the highest percentage inhibition of the fungus (70.78%) while 5 g at 144 hours incubation had the lowest percentage inhibition (6.69%). Also, at 48 hours of incubation, 25 g of M. lucida, A. conyzoides, and A. indica highly inhibited the growth of the fungus at 52.68%, 63.09%, and 62.33% respectively. This observation was not significantly different from the effect of 20 g extract of C. orbiculare. However, 73.68% of 25 g C. odorata had the highest growth inhibition effect against L. theobromae at 48 hours of incubation, while 6.25% of 5 g extract of the same plant had the lowest growth inhibition Table 2. Also, 25 g of M. lucida and A. conyzoides at 144 hours of incubation, as well as 25 g of A. indica at 72 hours incubation gave the highest growth inhibition of 64.71%, 48.09%, and 41.04%, respectively.

4. DISCUSSION

This study showed that aqueous extract of the C. odorata, M. lucida, A. conyzoides, and A. indica, as well asthe synthetic chemical (mancozeb), had significant effects on the radial growth of C. orbiculare and L. theobromae causing anthracnose and root-rot of cucumber. Several reports have documented the effects of plant extracts in controlling many phytopathogenic fungi (Abd-El-Khair & Haggag, 2007; Perez-Sanchez, Infante, Galvez, & Ubera, 2007). Plants generally produce many secondary metabolites which constitute an important source of microbicides, pesticides, and many pharmaceutical drugs (De Billerbeck, Roques, Bessière, Fonvieille, & Dargent, 2001). The efficacy of the inhibitory activities of selected plant extracts was enhanced with an increase in the concentration of extracts. This finding agrees with the report of Mares, Tosi, Poli, Andreotti, and Romagnoli (2004) that a higher concentration of antimicrobial substance showed an increase in growth inhibition. The observation was also in line with the earlier findings of Sidra and Bashir (2012) who reported that a higher concentration of plant extracts induced maximum inhibition on fungal growth. The minimum inhibitory concentration values of the plant extracts, in their report, against the test organism, showed that fungi vary widely in the degree of their susceptibility to antifungal agents.

The results revealed that all the plant extracts inhibited the growth of both cucumber pathogens. This could be as a result of the presence of phytochemical compounds such as mycotoxigenic proteins, steroids, terpenoids, anthraquinones, flavonoids, saponins, tannins, glycosides, glucosinulate, phenols, and alkaloids as reported by Gwa and Nwankiti (2017). C. odorata extract produced a higher inhibitory effect on radial growth of the fungal isolates in culture. The findings from the results revealed that all the plant extracts inhibited over 50% growth of C. orbiculare Chromolaena odorata, been the plant extract that gave the highest growth inhibition of the isolates, has also been reported by Ngane et al. (2006). They investigated theleaves and some of their fractions against yeasts and filamentous fungi through dilution methods on solid and liquid media, and observed extract from the plant possess significant antifungal properties. The authors reported that both extract and fractions can inhibit in vitro growth of Cryptococcus neoformans, Microsporum gypseum, Trichophyton mentagrophytes,and Trichophyton rubrum. This inhibitory property was attributed to the presence of biologically active constituents such as coumarins, flavonoids, phenols, tannins, and sterols through chemical analysis of the crude extract and its fractions (Onifade, 2000). As an allelopathic plant, extracts from all vegetative parts of Ageratum conyzoides including leaves have been reported to inhibit the growth of plant pathogens both in-vitro and in-vivo. The inhibitory effects observed in this study are also in line with the report of Sidra and Bashir (2012) who observed that the leaf extract of A. conyzoides inhibited the growth of Fusarium solani by 72% at the highest concentration of 6%. Extracts from this plant have also been documented to possess pharmacological and biocidal activity. It is evident from the present findings that the leaf extract of A. conyzoides possesses fungi toxic potentials worth exploiting against diseases caused by plant pathogens.  The leaves, bark, and root extract of Azadirachta indica has been reported to control Colletotrichum lindemuthianum of cowpea recording a 100% inhibition of spore germination and mycelia growth (Gwa & Nwankiti, 2017; Onifade, 2000).  Biu, Yusufu, and Rabo (2009) also revealed in their investigation, that the presence of anti-nutrients like saponins, tannins, glycosides, alkaloids, terpenes, and flavenoids in the aqueous extracts of the leaves of A. indica is responsible for its inhibitory potentials against mycelia growth of pathogens. Akinbode (2010) also reported that at higher concentrations, the mycelia growth of Curvularia lunata was inhibited by the extracts of Morinda lucida compared to the control plates.

Table-1. Antifungal activity of plantextracts on the growth inhibition (%) of Colletotrichum orbiculare of cucumber at various incubation durations.

Plant Extracts Concentrations (g/L)
Period of incubation (Hours)
48
72
96
120
144
Chromolaena odorata 5g
28.39c
15.47d
13.12d
7.90d
6.69e
10g
29.65c
29.64c
33.60c
26.39c
24.25d
15g
34.70c
36.86c
42.74c
30.52c
29.77d
20g
42.59c
37.37c
44.33c
44.88c
45.99c
25g
67.51b
69.85b
70.78b
67.68b
65.39b
Morinda lucida 5g
28.39c
33.76bc
41.75b
36.63b
31.44b
10g
35.96c
36.34bc
42.35b
40.22b
35.28b
15g
36.91c
38.91bc
43.74b
41.29b
35.95b
20g
40.06b
41.50b
45.73b
44.35b
38.63b
25g
52.68b
51.80b
46.92b
48.19b
39.30b
Ageratum conyzoides 5g
36.91c
31.19c
34.39c
23.70c
11.37d
10g
40.06c
34.28c
37.97c
25.49c
26.09c
15g
43.22c
36.08c
39.76c
39.50bc
35.95bc
20g
62.15b
54.90b
47.32b
48.83b
46.49b
25g
63.09b
57.47b
54.87b
51.89b
48.66b
Azadirachta indica 5g
30.11c
29.10c
31.32c
19.50c
10.17d
10g
38.23c
31.46c
32.11c
20.95c
20.99c
15g
43.12c
34.08c
36.15c
38.44b
31.00bc
20g
60.15b
51.00b
47.10b
42.93b
44.62b
25g
62.33b
55.40b
49.89b
48.21b
46.10b
Mancozeb 0.25g/100mL
100a
100a
100a
100a
100a

Note: Mean values with similar letter(s) down the column are not significantly different at 5% level of significance by Duncan’s Multiple Range Test (DMRT).

Table-2. Antifungal activity of plantextracts on the growth inhibition (%) of Lasiodiplodia theobromae of cucumber at various incubation durations.

Plant Extracts
Concentrations (g/L)
Period of incubation (Hours)
48
72
96
120
144
Chromolaena odorata
5g
7.42e
6.25d
5.85d
7.33c
11.46c
10g
26.56cd
7.92d
9.20d
7.62c
12.37c
15g
35.89c
12.92d
10.37d
7.63c
16.28bc
20g
44.26c
31.25c
38.63c
32.55b
26.17b
25g
73.68b
45.83b
55.33b
37.98b
27.08b
Morinda lucida
5g
28.42c
42.29c
38.37c
29.05c
20.59d
    Ageratum conyzoides
10g
39.79c
50.00b
40.14bc
43.97bc
43.09c
15g
50.13b
56.07b
50.80b
58.25b
54.85b
20g
52.71b
56.25b
58.61b
58.25b
58.38b
25g
56.07b
58.33b
60.92b
63.02b
64.71b
5g
20.94b
17.29c
8.17d
4.29d
1.03d
    Azadirachta indica
10g
27.65b
18.13c
9.95d
11.59cd
10.74ab
15g
32.04b
25.00bc
20.60bc
15.40c
12.35c
20g
37.99b
33.33b
31.97b
28.57b
25.44c
25g
37.99b
44.38b
41.39b
45.56b
48.09b
5g
15.48ab
26.46bc
20.60c
23.33bc
19.12c
    Mancozeb
10g
17.34c
26.46bc
21.85c
23.81bc
21.62c
15g
25.68b
29.79bc
24.16c
23.81bc
22.06c
20g
26.63b
32.71b
27.71c
25.40bc
23.09c
25g
31.89b
41.04b
40.85b
39.68b
36.32b
0.25g/100mL
100a
100a
100a
100a
100a

Note:  Mean values with similar letter(s) down the column are not significantly different at 5% level of significance by Duncan’s Multiple Range Test (DMRT).

5. CONCLUSION

In replacement of synthetic chemicals that pose various side effects on humans, animals, and the environment through their application on plants, the findings from this study suggest a new pathway in developing a potent, affordable fungicidal agent from plants like Chromolaena odorata, Morinda lucida, Ageratum conyzoides and Azadirachta indica. The results from the present study established that all the four plant extracts contained antifungal substances which are significantly toxic to Colletotrichum orbiculare and Lasiodiplodia theobromae isolated from the infected cucumber. These can be further investigated to develop to fungicides against phytopathogens. However, C. odorata, M. lucida, A. conyzoides, and A. indica extracts appeared to be very effective against C. orbiculare while C. odorata and M. lucida were effective against L. theobromae at all selected levels of concentrations and durations. The in vitro inhibitory activities suggest that these plants have the potentials to control root rot disease on cucumber; it is therefore suggested that their extracts could be further purified to isolate the active components and also screened for their in vivo effect on cucumber plant.

Funding: This study received no specific financial support.  

Competing Interests: The authors declare that they have no competing interests.

Acknowledgement: All authors contributed equally to the conception and design of the study.

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