Journal of Pesticides and Biofertilizers Iorungwa Gwa .J Pesticides and Biofertilizers http://doi.org/03.2018/1.10015
Research Article
Open Access
Fungitoxic Activities of Plant Extracts on Mycelial Growth inhibition of Fusarium Oxysporum Causal Agent Yam Tuber Rot in Zaki-Biam, Benue State, Nigeria Iorungwa Gwa Department of Crop Production and Protection, Faculty of Agriculture and Agricultural Technology, Federal University, Dutsin-Ma, PMB 5001, Katsina State, Nigeria
Corresponding Author: Iorungwa Gwa , Email:
[email protected] Phone: +234(0)8039357109. Received date: February 16, 2018; Accepted date : February 23, 2018; Published date: March 19, 2018. Citation this Article: Iorungwa Gwa. Fungitoxic Activities of Plant Extracts on Mycelial Growth Inhibition of Fusarium Oxysporum Causal Agent Yam Tuber Rot in Zaki-Biam, Benue State, Nigeria, j pesticides and bio fertilizers, Doi : http;//doi.org/03.2018/1.100015.
Copyright: © 2018 Iorungwa Gwa. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract: Hot water extracts of five fungicidal plants (Piper guineense, Zingiber officinale, Azadirachta indica, Carica papaya and Nicotiana tabacum) and a synthetic fungicide (mancozeb) were tested for in vitro inhibitory activities on Fusarium oxysporum mycelial growth, the causal agent of yam tuber dry rot in storage using three different concentrations of plant extracts (30 g/L, 60 g/L and 90 g/L) and synthetic fungicide; mancozeb (4 g/L, 8 g/L and 12 g/L). The experiments were conducted at Advanced Plant Pathology Laboratory, Federal University of Agriculture, Makurdi, Nigeria. 5 mL of each concentration of the extract and chemical were separately amended in 15 mL of potato dextrose agar (PDA) in Petri dish and F. oxysporum was inoculated and incubated for 120 hours to determine the levels of effectiveness of the fungicides. The result showed that all the plant extracts were able to inhibit the mycelial growth of the pathogen with concentration III having the highest inhibitory effect in all the extracts. The three different concentrations of P. guineense were the best in activity followed by Z. officinale while the least effective extract was N. tabacum. There was a 100% inhibition when mancozeb was used irrespective of the levels of concentrations. Treatment of the test plant extracts significantly (P≤0.05) reduced mycelial growth of F. oxysporum in vitro. The concentrations of 60 g/L and 90 g/L of plant extracts and 4 g/L of mancozeb consistently gave the highest percentage growth inhibition of the pathogen and were considered the best in controlling the pathogen. This study therefore, revealed that P. guineense, Z. officinale, A. indica, C. papaya and N. tabacum were able to arrest the growth of F. oxysporum, the rot-causing fungus of white yam. These extracts will therefore, serve as good plant fungicides in protecting yam tubers against rot causing fungi in storage Keywords: Fungicidal, Plant extracts, Fusarium oxysporum, inhibition, yam, Zaki-Biam . Introduction Yam production has always been reported to be on a high scale throughout the world and mostly in West Africa [1]. The greater part of world yam production (over 90%) is derived from West Africa. In 2008, Nigeria was the largest producer of yam in the world, producing 35.02 million metric tonnes [2]. Although it is grown widely in Nigeria, the area where it is grown most is Benue State (land area of 802,295 km²) one of the states in Benue valley of Nigeria. Auctores Publishing – Volume1-10015 www.auctoresonline.org
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In this state especially among Tiv people, the size of the yam farm or the tonnage of yams produced becomes the social status of that farmer. Because of high level of yam production in the State of Benue, Benue State is crowned as the Nigerian Food Basket. Zaki-Biam in Benue State is the largest producer with the largest market for yam in the world [3]. Rot is a major factor limiting the Post-harvest life of yams besides lack of research for development and capacity building in yambased researches.
Journal of Pesticides and Biofertilizers
Studies conducted by [3-6] reported that 50% of the yam tubers produced and harvested in Nigeria are lost to diseases in storage while [2] estimated an average of over 25% are lost to diseases and pest. The major fungi organisms causing rots of yam in Nigeria includes: Aspergillus flavus, Aspergillus niger, Botryodiplodia theobromae, Collectotrichum spp, Fusarium oxysporum, Fusarium solani, Geotrichum candidum, Penicillium chrysogenum, Pennicillium digitatum [7-10]. These organisms reduce the quantity of yam produced and also the quality [11]. Chemicals have proved helpful in the control of yam diseases especially when the tubers are already attacked by pathogens [12]. The major problems of chemicals are that they posed a great challenge to the ecosystem and also frequent uses of chemicals predispose target organisms to resistance. The use of pesticides of plant origin has been recommended by researchers as alternatives to synthetic chemicals so as to prevent the environmental risks associated with the use of synthetic chemicals [13, 14]. The study was therefore aimed at evaluating the in vitro fungitoxic activities of some plant extracts in the control of yam tuber dry rots caused by F. oxysporum in vitro.
Preparation of Plant extracts
Materials And Methods
The approach of [21] was used to evaluate the fungitoxic effect of the plant extracts and the chemical fungicide on fungal mycelia growth by creating four equal sections on each plate. This involves drawing two perpendicular lines at the bottom of the plate. The point of intersection indicates the centre of the plates. These were done before dispensing PDA into each of the plates. The prepared medium was poured into sterilized Petri dishes and 5 ML of each plant extracts and chemical fungicide at the different level of concentrations were poured into Petri dishes containing 15 ML of potato dextrose agar media separately [22], mixed well and allowed to solidify. The solidified medium was inoculated centrally at the point of intersection of the two perpendicular lines drawn at the bottom of the plate with discs (5mm diameter) which were obtained from one-week-old cultures of the test fungus. The control experiments had 5ML of distilled water added to 15 ML of PDA in place of plant extracts and chemical fungicide respectively; the treatments and control were replicated three times and incubated for 120
Experimental site The experiment was conducted at the Advanced Plant Pathology Laboratory, Federal University of Agriculture, Makurdi, Nigeria. Collection of diseased yam tubers Rotted yam tubers of white yam varieties (Dioscorea rotundata) showing various diseased symptoms of rots were collected from yam farmers from various storage barns in Zaki-Biam market, Benue State, Nigeria which lies between longitudes 9o 25' and 9o 28'E, and latitude 7o 32ʹ and 70 35′N respectively. The rotted yam tubers were packaged in sterile polyethylene bags, taken to the laboratory for isolation and identification of pathogens. The tubers were protected using wire mesh to prevent rodent attack [14]. Potato Dextrose Agar (PDA) was the medium used. Test fungus for this study was F. oxysporum. Isolation and identification of F. oxysporum Sections of the yam tubers were cut under aseptic condition into small bits of approximately 2x2mm from yam tubers infected with rot at inter-phase between the healthy and rotten portions. The cut tissues were soaked in 5% sodium hypochlorite for 2 minutes for surface sterilization and then rinsed in four successive changes of sterile distilled water as reported by [15]. The infected tissues were later picked onto sterile filter paper using a sterile forceps and then wrapped with filter paper for 2–3 minutes. The dried infected tissues were placed onto several prepared sterile plates of acidified potato dextrose agar (PDA) and the plates were incubated at ambient room temperature (30±5°C) for 7 days. The fungal colonies grown on the incubated plates were sub-cultured into fresh separate sterile acidified PDA plates and incubated to obtain pure cultures of pathogens. Macroscopic and microscopic examination and morphological characteristics and identification were made and compared with existing authorities [16, 17] Pathogenicity test Healthy yam tubers were washed with tap water, rinsed with distilled water and surface sterilized with 5% sodium hypochlorite. Cylindrical discs were removed from the tubers with a sterile 5 mm cork borer. A disc of a five days old culture of F. oxysporum was transferred into holes created in the tubers; petroleum jelly was used to completely seal the holes. The same procedure was used for the control except that discs of uninoculated PDA were placed in the holes created in the tubers [18]. After incubation period of 14 days at ambient room temperature (30±5°C) the tubers were examined for infection and disease development. Auctores Publishing – Volume1-10015 www.auctoresonline.org
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The method of [19, 20] were used with some modifications. Seeds of Piper guineense (Black Pepper), Rhizomes of Zingiber officinale (Ginger), leaves of Azadirachta indica (Neem), leaves of Carica papaya (Pawpaw) and leaves of Nicotiana tabacum (Tobacco) were washed thoroughly with cold running tab water, air-dried and separately grounded into fine powder using a mortar. Hot water (100oC) extraction was obtained by adding 30g, 60g and 90g of the powder of each plant extracts at the different level of concentrations to 1litre of sterile distilled water separately in 1000 ml Pyrex flask. These were left for 24 hours and subsequently filtered through four fold of sterile cheese cloth. The filtrates obtained were used as the plant extracts in the experiment. Mancozeb was prepared in sterile distilled water at 4 g/L, 8 g/L and 12 g/L concentrations respectively. The efficacies of the aqueous plant extracts and chemical fungicide were tested in vitro for their fungicidal activity against tuber rot of yam (Dioscorea rotundata) caused by F. oxysporum. Effect of the plant extracts on F. oxysporum mycelial growth
o
hours at ambient room temperature (30 ±5 C). Measurements of growth as radius of a growing fungal colony were undertaken at intervals of twenty four hours for 120 hours using a transparent ruler. The absence of growth in any of the plates was indicative of the potency of the extract and the chemical fungicide against the test fungus. Fungitoxicity was determined as percent growth inhibition (PGI) according to the method described by [23]. 𝑅 –𝑅1 𝑃𝐺𝐼 (%) = × 100 𝑅 Where, PGI = Percent Growth Inhibition R = the distance (measured in mm) from the point of inoculation to the colony margin in control plate, R1 = the distance of fungal growth from the point of inoculation to the colony margin in treated plate.
Experimental Design and Data Analysis The experimental design was completely Randomized Design (CRD) with three replications as described by [24]. Test of variance was calculated using Analysis of variance (ANOVA) and statistical F-tests were evaluated at P≤ 0.05. Differences among treatment means for each measured parameter were further separated using fishers least significance difference (LSD) to determine levels of significance according to [25]. GenStat Discovery Edition 12 was used for ANOVA and means separation, Minitab Release 17 for descriptive statistics and Graph Pad Prism 6 for trend graphs.
Results Fusarium oxysporum was isolated and identified as one of the major yam tuber rot organisms in the study area. Colony characteristics growth on PDA was rapid covering the entire plate after 120 hours of incubation. There was white aerial mycelium colour (Figure 1A).
Journal of Pesticides and Biofertilizers
The pathogenicity test carried out revealed that F. oxysporum inoculated into the yam tubers was able to induce rot; this was probably due to the ability of the fungus to utilize the nutrients of the yam tubers as substrates for growth and development (Figure 1B). The uninoculated yam tubers however, showed no growth (Figure 1C).
oxysporum but none was able to completely inhibit its radial growth except the synthetic chemical mancozeb which completely inhibited the growth of the pathogen irrespective of the concentration (Table 1). The rate of growth of the test fungus was influenced by the type of extract used which decreased with increase in concentrations of the extracts. There were significant differences (P≤ 0.05) among the concentrations of the test plants on mycelial radial growths of F. oxysporum throughout the period of incubation (Table 1).Period of incubation has no significant difference for concentration I and III of Z. officinale; concentration III of C. papaya and concentration II of A. indica. The synthetic fungicide, mancozeb which showed 100% growth inhibition was considered more effective compared with the plant extracts and also showed significant difference with the plant extracts at all the levels of concentrations and throughout the period of incubation (Table 1).
A B C Figure 1: Pure culture of F. oxysporum growing on Potato Dextrose
Table 1and 2 showed variations in the mycelial growth of the test fungus. The rate of growth was influenced by the type of extract used and this also increased with increase in concentrations of the extracts. There was a significant difference in statistical test between concentration I and III (P≤ 0.05) on mycelial radial growths of F. oxysporum when P. guineense extract was used throughout the period of incubation. There was however, significant difference between concentration I, II and III of P. guineense. Period of incubation has no significant difference for concentration I, II and III of Z. officinale, C. papaya and concentration II and III of A. indica. The synthetic fungicide, mancozeb which showed the highest percentage growth inhibition was considered more effective than the plant extracts and also showed significant difference with the plant extracts at all the levels of concentrations and throughout the period of incubation (Table 1 and 2).
Agar (A), Pathogenicity test showing rot caused by F. oxysporum (B) and control without F. oxysporum (C).
In-vitro evaluation of the effect of plant extracts and fungicide on the mycelial growth of F. oxysporum The effect of different concentrations of plant extracts and fungicide on mycelial radial growth of F. oxysporum shown in Table 1 indicated that Z. officinale and P. guineense showed fungicidal properties higher than A. indica, C. papaya and N. tabacum at all the concentration levels. The tested plant extracts inhibited the growth of F.
Plant Extract Piper guineense Zingiber officinale Azadiracta indica Carica papaya Nicotiana tabacum
Mancozeb
Concentration (g/L) Conc I (30)
Period of Incubation (Hours) and Percentage Growth Inhibition (%) 24 48 72 96 120 36.11±7.35b 59.40±4.93a 53.50±10.40ab 59.79±3.85a 48.60±2.94ab
P-Value 0.04
Conc II (60) Conc III (90) Conc I (30) Conc II (60) Conc III (90) Conc I (30) Conc II (60) Conc III (90) Conc I (30) Conc II (60) Conc III (90)
61.10±13.90 83.33±8.33a 52.80±12.10 52.80±12.10 69.40±19.40 25.00±14.40b 44.44±5.58b 69.40±19.40 33.30±16.70 58.30±30.00 69.40±19.40
69.27±5.36 75.93±3.38ab 59.56±1.29 63.98±5.25 74.05±3.72 56.81±5.47a 61.94±1.25a 71.57±2.98 45.23±1.19 62.26±9.23 69.45±4.98
63.70±4.18 70.46±1.91ab 52.85±6.6.98 63.70±4.16 57.70±3.86 49.56±1.56a 51.52±5.27ab 59.98±5.05 34.70±14.10 53.56±5.31 55.70±8.26
66.15±3.05 71.57±1.72ab 57.70±3.89 63.12±2.82 67.19±3.65 51.43±2.77a 57.77±2.23a 65.11±2.81 49.36±2.59 58.81±2.79 64.17±1.27
59.87±4.81 62.86±1.45b 55.95±8.15 58.18±3.28 63.84±1.40 41.95±1.58ab 53.39±1.76ab 62.88±1.44 40.84±4.26 50.53±1.71 58.04±2.87
0.89ns 0.04 0.95ns 0.74ns 0.92ns 0.04 0.04 0.89ns 0.76ns 0.97ns 0.79ns
Conc I (30)
27.78±2.78b
38.06±1.25ab
38.83±4.79a
42.94±4.44a
42.88±2.92a
0.04
Conc II (60)
52.80±12.10
44.88±4.41
42.89±8.13
55.82±0.64
50.45±1.23
0.65ns
Conc III (90)
61.10±20.00
59.54±5.95
53.74±3.87
64.16±3.86
60.12±3.79
0.95ns
Conc I (4)
100.00±0.00
100.00±0.00
100.00±0.00
100.00±0.00
100.00±0.00
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Conc II (8) Conc III (12)
100.00±0.00 100.00±0.00
100.00±0.00 100.00±0.00
100.00±0.00 100.00±0.00
100.00±0.00 100.00±0.00
100.00±0.00 100.00±0.00
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Table 1: In vitro effect of different filtrate concentrations of some plant extracts and chemical fungicide at different concentrations on Percentage Growth Inhibition of Fusarium oxysporum after 120hours of incubation.
Means on the same row (for each Plant Extract) with the different superscript are statistically significant (p
