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Research Article | DOI: https://doi.org/
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Citation:
Copyright: © 10.31579/2578-8825/013
Received: 30 November -0001 | Accepted: 01 January 1970 | Published: 19 March 2018
Keywords: Fungicidal, Plant extracts, Fusarium oxysporum, inhibition, yam, Zaki-Biam
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 thatP. 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
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. 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 yam-based researches. 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. oxysporumin vitro.
Materials And Methods
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 9o25' 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]
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.
Preparation of Plant extracts
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 Nicotianatabacum (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
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 mediaseparately [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 hours at ambient room temperature (30 ±5oC). 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]:
PGI %= R –R1 R ×100
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.
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<0.05) by period of incubation.
Means on the same column (for each Plant Extract) with the different subscript are statistically significant (p<0.05) by concentration, ns=not significant
Mean percentage growth inhibition of three concentrations of plant extracts (30g/L, 60g/L and 90g/L) and three concentrations of mancozeb (4g/L, 8g/L and 12g/L) on mycelial growth inhibition of F. oxysporum throughout the period of incubation showed a statistical significant (P≤ 0.05) among plant extracts from 24 hours to 120 hours period of incubation. P. guineense showed the highest level of inhibitions among the plant extracts throughout the period of incubation followed byZ. officinale with least percentage growth inhibition by N. tabacum (figure 2). Generally, the effectiveness of the extracts increased with increase in concentration irrespective of the type of extract used. There was however, no significant difference in the performance of mancozeb in the inhibition of mycelial growth of F. oxysporum irrespective of concentration (Table 3).
Azadiracta indica
Table 2: In vitroPercentage Growth Inhibition ofFusarium oxysporum by some plant extracts and chemical fungicide at different concentrations after 120hours of incubation
Means on the same column (for each concentration) with different superscript are statistically significant (p<0.05). (Conc I=30 g/L of Plant extract, 4 g/L of Mancozeb; Conc II = 60 g/L of Plant extract, 8 g/L of Mancozeb; Conc III = 90 g/L of Plant extract, 12 g/L of Mancozeb)
Mean percentage growth inhibition of F. oxysporum after 120 hours of incubation showed an increase in the performance of the extracts from the lowest concentration to the highest concentration. When concentration I (30 g/L plant extract and 4 g/L mancozeb) was used the percentage growth inhibition was highest in Z. officinale followed by Piper nigrum with the least percentage growth inhibition recorded from N. tabacum. The extracts increased their activities in reducing mycelial growth of F. oxysporumwhen concentration II (60 g/L plant extract and 8 g/L mancozeb) was used with the highest percentage growth inhibition from P. guineense followed by Z. officinale with the least value recorded against N. tabacum. The trend continued the same way with concentration III (90 g/L plant extract and 12 g/L mancozeb). Fungitoxicity was highest with P. guineense followed by Z. officinale while N. tabacum still maintained the least in activity. There was a significant difference (P≤ 0.05) on the activities of all the extracts on F. oxysporum growth at different concentrations for the period of incubation. There was however, no significant difference in the performance of mancozeb in the inhibition of mycelial growth of F. oxysporumirrespective of level of concentrations (Table 3).
Plant Extract
Concentrations (g/L) and Percentage Growth Inhibition (%)
Table 3: Mean Percentage Growth Inhibition of F. oxysporum by some plant extracts and chemical fungicide at different concentrations after 120 hours of incubation
Means on the same column with the different superscript are statistically significant (p<0.05). (Conc I=30 g/L of Plant extract, 4 g/l of Mancozeb; conc II = 60 g/L of Plant extract, 8 g/L of Mancozeb; Conc = 90 g/L of Plant extract, 12 g/L of Mancozeb)
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). 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).
Period of Incubation (Hours) and Percentage Growth Inhibition (%)
Period of Incubation (Hours) and Percentage Growth Inhibition (%)
Concentration I
Nicotiana tabacum
Concentration II
Concentration III
The test pathogen from the rotted yam samples was identified as F. oxysporum, one of the organisms causing yam dry rot.Previous work had implicated F. oxysporum as one of the yam tuber rot-causing organisms [26, 27]. The extracts showed their potency to inhibit the growth of the tested organism in culture. This result is similar to the findings of previously done works on the use of plant extracts in the control of fungal rots [19, 27-29]. The results of filtrate concentrations of plant extracts on PDA medium showed that P. guineense, Z. officinale, A. indica, C. papaya and N. tabacum extracts were all fungitoxic to F. oxysporum at all concentrations but none of them were 100% efficacious in inhibiting radial mycelial growth of F. oxysporum. However, the effectiveness was observed to be higher in P. guineense, Z. officinale extracts compared withA. indica, C. papaya and N tabacum extracts. This could probably be as a result of higher concentrations of the active principles in P. guineense, Z. officinale compared with A. indica, C. papaya and N. tabacum. The availability of active principles in the extracting solvent is determined by factors like age of plant and method of extraction [30, 31]. Antimicrobial activity of different extracts increased as the concentration increased. This result is in tandem with earlier investigations by [32, 33]. The actions of the antifungal substances present in the plant extracts were fungistatic at lower concentrations but became fungicidal at higher concentrations as reported by [34]. The presence of fungicidal compounds such as piperine in P. guineense, gingerol in Z. officinale, azadirachtin in A. indica, papain in C. papaya and nicotine in N. tabacum caused the inhibition of mycelial growth of F. oxysporum in vitro agreed with the reports of other investigators [29, 35, 36]. Report by [19] showed that the growth of F. oxysporum mycelial was inhibited using cold extracts of N. tabacum, A. inndica, Aloe barbadensis, Tridax precubens and Carica papaya. The result also agreed with the work of [37] that controls yam tuber rot pathogens using leaf extracts of Xylopia aethiopica and Zingiber officinale. This result further confirms the findings of [27]who also reported the control of C. lindemuthianum using neem seed, fruit, leaf; bark and root extract, recording a 100% inhibition of spore germination and mycelial growth. Similar results were obtained by [38, 39] who used P. nigrum, Z. officinale, A. indica, C. papaya and N. tabacum and inhibited the mycelial growth of Aspergillus flavus and Colletotrichumspecies in vitro respectively.
Assessment of the effect of mancozeb on F. oxysporum mycelial showed that increase in the concentration of the chemical does not affect growth inhibitions as the test fungus had already attained the highest level of inhibition at the lowest concentration possible. This result agreed with the work earlier on done by [40] who found out that mancozeb consistently gave 100% inhibition (at concentrations of 250ppm, 500ppm and 1000ppm) of germination of conidia of Cercospora contraria and Didymosphaeria donacina which caused leaf spot diseases of cluster yam (Dioscorea dumetorum).
The research has demonstrated that all the plant extracts contained antifungal properties to control F. oxyysporum, one of the major post harvest yam rots causing pathogens. This is an alternative way of reducing the economic losses associated with rots causing pathogens of yam in storage. This will help to prevent the adverse effect caused by synthetic chemicals to our environment.
Conflict Of Interest Disclosure
The authors declare that there is no conflict of interest regarding the publication of this paper.
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