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Open Access Journal of Microbiology & Biotechnology Research Article 28 min read

Antimicrobial Activity of Five Plant Extracts and Synthetic Fungicide in the Management of Postharvest Pathogens of Yam (Dioscorea Rotundata Poir) in Storage

Gwa VI*, Nwankiti AO and Hamzat OTH
* Corresponding author
ISSN: 2576-7771  10.23880/oajmb-16000128  Received: July 07, 2018  Published: August 17, 2018
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Keywords
Antimicrobial Aspergillus ochraceus Inhibition Mycelial Plant extracts Post harvest
Abstract

Crude extracts leaves of Azadirachta indica A. Juss. (neem), Nicotiana tabacum Linn. (Tobacco), rhizomes of Zingiber officinale Rosc. (Ginger), leaves of Carica papaya Lam. (pawpaw) and seeds of Piper guineense Schumach. (Black pepper) were tested at 30 g/L, 60 g/L and 90 g/L as well as mancozeb at 4 g/L, 8 g/L and 12 g/L concentrations against Aspergillus ochraceus in vitro. Rotted yam tubers were collected from farmers’ barns forth nightly for four times. Treatments were replicated three times and completely randomized. Data were subjected to analysis of variance (ANOVA) and means were separated using Fisher’s least significance difference (LSD). Mycelial growth of A. ochraceus was inhibited by these extracts. Percentage growth inhibitions at 30 g/L ranged from 40.19% (N. tabacum) to 51.95% (P. guineense) while percentage growth inhibitions at 60 g/L ranged between 57.47% (N. tabacum) and 70.56% (P. guineense). The highest inhibitions were recorded at 90 g/L with a range between 66.22% (A. indica) and 74.15% (P. guineense). Mancozeb was found to be the best in inhibiting the mycelial growth of A. ochraceus in culture irrespective of concentration and duration of incubation with 100% growth inhibition. The extracts and mancozeb were sprayed on yam tubers before storage for five months. Stored tubers showed a decay reduction index of 1.00 (mancozeb), 0.93 (A. indica) and 0.83 (P. guineense and Z. officinale) in the first year of storage indicating a reduction in rot by 100%, 93% and 83%, respectively. The extracts were comparatively less effective in the second year of storage. It is therefore, concluded that plant extracts could be used in management of rot causing organisms of yam in storage since they are environmentally safe, cheap and easily available compared with synthetic chemicals.

Introduction

Yam (D. rotundata) production and consumption is mostly in West Africa, East Africa, the Caribbean, South America, India and South East Asia [1, 2]. Nigeria has the highest production record of 38.92 million metric tonnes of yam tubers annually [3, 4]. Production of yam is constrained by several factors such as high cost of production, pests and diseases. Pathogenic micro- organisms including bacteria, fungi and viruses attack yams at all stages of growth and also during storage of tubers [5, 6, 7, 8, 9, 10]. These constraints are responsible for reduction in growth, tuber formation and development as well as tuber quality during storage [11]. Though a yam tuber naturally has a periderm that microorganisms cannot breach, it is easily wounded by other pest organisms such as insects, rodents, nematodes and even man during weeding, harvesting and post-harvest handling. These wounds serve as entering points to these rot inciting microorganisms [12]. Losses due to rot organisms were estimated to be as low as 10-15% in the first three months of storage. Okigbo, Arinze and Okigbo, et al. [13, 14, 15] estimated losses to diseases in storage to be as high as 50% of the yam tubers produced in Nigeria. According to Sadiku and Sadiku [16] rot caused by pathogenic organisms may be exacerbated due to challenges in climate change. Various types of synthetic fungicides have been used to control different types of plant diseases in different crops. The major disadvantages are the development of resistance to many of the currently used chemicals in circulation, pollution of soil, ground and surface water as well as the atmosphere [17, 18]. Biological control based on use of plant products as well as use of antagonists is the popular alternative to the use of synthetic fungicide control [19, 20, 21, 22, 23]. Some plants are known to synthesize phytochemical compounds with antimicrobial potencies and are used successfully in the control of diseases in humans and crops such as yam, cassava, tomato, cowpea, rice, etc. [10, 24, 25]. The advantages of using these natural plant products are enormous and include; little or no mammary toxicity, local availability, biodegradability and simple preparation procedures [9, 26]. The aim of the study was therefore, to evaluate the antimicrobial activities of some selected plant extracts in the in vitro control of A. ochraceus and in vivo control of some pathogenic organisms causing rot of yam tubers in storage and recommend the use of these plant extracts to farmers.

Materials and Methods

Sample Collection of Diseased Yam Tubers

Decayed yam tubers (D. rotundata) with varying degrees of rots showing different kinds of symptoms from Tor-Donga, Benue State, Nigeria were collected from different farmers’ barns. The collected tubers were packaged in sterile polyethylene bags in order to prevent them from further attack by insects and pathogens and were thereafter taken to the Advanced Plant Pathology Laboratory, Federal University of Agriculture, Makurdi, Nigeria for isolation and identification. In the laboratory, the samples were protected from rodent attack using wire mesh. The medium used for growing fungal organisms was Potato Dextrose Agar (PDA) which was prepared according to manufacturer’s recommendation.

Isolation of Aspergillus Ochraceus

Infected yam tubers showing various degrees of rot symptoms were washed in running tape water before cutting at interphase between rotted and healthy tissues. Cut tissues were surface sterilized in 5% Sodium hypochlorite (commercial bleach) solution for approximately 2 minutes. Tissues were thereafter removed and rinsed in four successive changes of sterile distilled water. The pieces were then placed on sterile what man No.42 filter paper (Sigma-Aldrich) in the laminar Air flow cabinet to dry for 2 minutes before inoculation.

Identification of A. ochraceus

Dried infected tissues were later picked from the sterile filter paper using sterile forceps and were aseptically platted in sterilized Petri plates containing solidified freshly prepared potato dextrose agar (PDA) medium. The plates were incubated at ambient room temperature (30±5°C) for five days to allow for adequate growth of fungal mycelial. Different fungal colonies were seen on the plates, from which A. ochraceus was identified, purified and multiplied on PDA. Identification was on the basis of growth using cultural, morphological and microscopic characteristics and the characteristics were compared with existing authorities [27, 28].

Pathogenicity Test of A. ochraceus

The method of Gwa and Nwankiti [10] were adopted for pathogenicity test of A. ochraceus. Healthy yam tubers were obtained from same location where rotted yam were collected, they were washed in running tap water, sterilized with 5% Sodium hypochlorite solution for 30 seconds and rinsed in four successive changes of sterile distilled water. A sterile 5 mm cork borer (sterilized by flaming) was punched into the healthy looking yam tubers to a depth of 4 mm and the bored tissues were removed. A 5 mm diameter disc from the pure culture of A. ochraceus was cut and replaced in the holes created. Same procedure was used for the control experiment except that sterile agar discs were used instead of the inoculum obtained from A. ochraceus in the holes created in the tubers [22]. The remaining parts of the holes were completely sealed with Petroleum jelly to prevent contamination by other pathogenic organisms. Treatments with A. ochraceus tubers were replicated three times and the yam tubers were incubated at ambient room temperature (30±5°C) under sterile condition for 14 days for growth to establish. When growths were fully established, the inoculated yam tubers were cut transversely at point of inoculation to determine the extent of infection and disease development. Disease symptoms that developed on the artificially inoculated yam tubers with A. ochraceus after the incubation period were compared with those naturally infected tubers initially collected from farmers’ barns. Re-isolation of A. ochraceus from the inoculated diseased yam tubers were done and cultured on PDA plates. The characteristic culture of the tested fungus obtained was compared with the culture initially collected from the naturally infected tubers.

Preparation of Plant Extracts

The methods of Gwa and akombo and Gwa, et al_. [9, 22] were used. 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 tap water, air- dried and separately ground into fine powder using pestle and mortar. Measurement of 30 g, 60 g and 90 g of the powder of each plant extracts was added to 1litre of sterile hot (100oC) distilled water separately in 1500 Ml Pyrex flask. The mixtures were left for 24 hours before filtration using four-fold sterile cheese cloth to give concentrations of 30 g/L, 60 g/L and 90 g/L, respectively. The filtrates obtained were used as plant extracts in the experiment. The synthetic chemical; mancozeb, was prepared by dissolving 4 g, 8 g and 12 g separately in one litre of cold sterile distilled water to obtain concentrations of 4 g/L, 8 g/L and 12 g/L, respectively. The potencies of the plant extracts as well as the synthetic fungicide were compared for their in vitro fungicidal activity in reducing mycelial growth of A. ochraceus and in vivo control of yam pathogens in storage.

In vitro Antifungal Activity of Plant Extracts on the Mycelial Growth of A. ochraceus

The antifungal in vitro assays were carried out following the method described by Amadioha and Obi, [29]. It involves creating four equal sections on each plate by drawing two perpendicular lines at the bottom of the plate. The intersection of the two lines indicates the centre of the plates. This was 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 their respective concentrations were poured into Petri dishes containing 15 ml of the medium separately [30] 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 5 mm diameter discs which was obtained from one-week-old cultures of A. ochraceus grown on PDA plates as inoculum [10, 31]. The treatments were replicated three times for each of the concentrations. The procedure was the same in the control experiments except that 5 ml of sterile distilled water was added to PDA instead of plant extracts. The treatments and control experiments were completely randomized [32] and incubated at ambient room oC) for 120 hours. Measurement of mycelial radial growth was done and fungitoxicity was calculated as percentage growth inhibition of A. ochraceus using the formula described by Korsten and De Jager.

temperature (30 ±5 𝑃𝐺𝐼 % = 𝑅 – 𝑅1

𝑅 × 100

Where, PGI = Percentage 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. The efficacies of the aqueous plant extracts and the synthetic fungicide that were found to be effective against A. ochraceus in vitro were used to control pathogens of yam tubers in storage for five months.

Effectiveness of Some Plant Extracts and Chemical Fungicide in Controlling Yam Tuber Rot Pathogens in Storage

The efficacy of three plants extracts namely: seeds of P. guineense, rhizomes of Z. officinale and leaves of A. indica and chemical fungicide (mancozeb) that were found to posses’ more fungicidal properties in vitro were tested for efficacy in controlling yam tuber rot pathogens in vivo at different level of concentrations. Plant extracts were prepared as described above and Ogoja cultivar of white yam tubers were each sprayed with three plant extracts at concentrations of 30 g/L, 60g/L and 90 g/L, respectively using a hand sprayer. The synthetic chemical, mancozeb was applied at a concentration of 4 g/L respectively since there were no differences in culture in percentage growth inhibition of A. ochraceus using 8 g/L and 12 g/L. After spraying, the tubers were allowed to dry after which tubers were stored for five months. Three tubers formed a treatment; this was replicated three times bringing the total to 9 tubers per treatment. There were 11 treatments (three plant extracts at three concentrations each, mancozeb and control). A total of 99 tubers of Ogoja yam were used for the experiment. The treatments were completely randomized and control was set up for each cultivar in which sterile distilled water was sprayed on the yam tubers and allowed to dry (no plant extract or chemical applied). Data on the potency of the extracts and chemical fungicide in controlling rot causing pathogens during storage were collected at monthly interval for five months. The numbers of Fungal organisms identified in Tor-Donga were Fusarium moniliforme, F. oxysporum Botryodiplodia theobromae, Aspergillus flavus, A. niger, A. ochraceus, Penicillium purpurogenum and Pestalotia sp. Cultural and morphological characteristics of A. ochraceus on PDA shows rapid and fast growth. There was powdery brown colour almost covering the plate within 7 days of incubation (Figure 1: Plate 1). Microscopic examination shows that each conidiophore ended in a terminal enlarged spherical swellings (Figure 1: Plate 2).

Figure 1: Plate 1). Microscopic examination shows that each conidiophore ended in a terminal enlarged spherical swellings (Figure 1: Plate 2).
Click to enlarge
Figure 1: Plate 1). Microscopic examination shows that each conidiophore ended in a terminal enlarged spherical swellings (Figure 1: Plate 2).

unrotten and rotten tubers in each treatment were recorded. The effectiveness of the concentrations of plant extracts and chemical fungicide in controlling yam tuber rot pathogens in storage were evaluated. The Decay Reduction Index [33] defined below, was calculated as a measure of the effectiveness of each plant extract and chemical fungicide in controlling yam tuber rot pathogens in storage at different concentrations after final data collection as:

𝐷𝑒𝑐𝑎𝑦 𝑅𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝐼𝑛𝑑𝑒𝑥 (𝐷𝑅𝐼)

= % 𝑑𝑒𝑐𝑎𝑦 𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙−% 𝑑𝑒𝑐𝑎𝑦 𝑖𝑛 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑡𝑢𝑏𝑒𝑟𝑠 % 𝑑𝑒𝑐𝑎𝑦 𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

Statistical Analysis

Analysis of variance (ANOVA) using Genstat Discovery Edition 12 for ANOVA and means separation and Graph Pad Prism 6 for trend graphs were used. Statistical F-tests were evaluated at P≤ 0.05 using Fisher’s least significant differences (FLSD) [34].

Results

Pathogenicity Test

Result of pathogenicity test conducted on Pepa white yam using the inoculum collected from A. ochraceus shows that the pathogen was able to induce rot in the healthy looking yam tubers 14 days after inoculation (Figure 2: Plate 3). Healthy Pepa yam tubers that were not inoculated with the test fungus showed no symptoms of rot in the bored yam tubers (Figure 2: Plate 4).

Figure 2: Plate 3). Healthy _Pepa_ yam tubers that were not inoculated with the test fungus showed no symptoms of rot in the bored yam tubers (Figure 2: Plate 4).
Click to enlarge
Figure 2: Plate 3). Healthy Pepa yam tubers that were not inoculated with the test fungus showed no symptoms of rot in the bored yam tubers (Figure 2: Plate 4).

only Z. officinale and N. tabacum were not significantly different (P≤ 0.05) across duration of incubation while all the plant extracts significantly inhibited the mycelial growth of A. ochraceus at concentration III (90 g/L) respectively. The results further revealed that there were significant differences (P≤ 0.05) among plant extracts for each of the concentration levels tested throughout the period of incubation (Table 2). Mean percentage growth inhibition of A. ochraceus after 120 hours of incubation showed that P. guineense and Z. officinale were more effective at each level of concentration; this was closely followed by A. indica at concentration I and II while N. tabacum was third in activity at concentration III (Table 2). Mean percentage growth inhibition of A. ochraceus after 120 hours of incubation showed significant differences (P≤ 0.05) among plant extracts for each concentration (Table 2). Mean percentage growth inhibition of three concentrations (30 g/L, 60 g/L and 90g/L) of each plant extracts on A. ochraceus throughout the period of incubation revealed that the plant extracts were more effective at the beginning initial period of incubation compared with the later periods (Figure 3).

In vitro Fungitoxic Effect of Plant Extracts and Fungicide on the Mycelial Growth Inhibition of A. ochraceus

Table 1 shows that P. guineense, Z. officinale, A. indica, C. papaya and N. tabacum extracts possess fungicidal properties at different concentration levels against A. ochraceus in vitro. However, only mancozeb gave 100% inhibition of mycelial growth of A. ochraceus indicating that only the synthetic fungicide completely inhibited the growth of the pathogen. All the plant extracts were able to reduce growth of the test fungus throughout the period of incubation irrespective of the level of concentration used and duration of incubation. Though the potency of the extracts decreased with increase in the period of incubation; the test fungus was effectively controlled throughout the test period. P. guineense and Z. officinale were considered better extracts than A. indica, C. papaya and N. tabacum at all the level of concentrations after 72 hours. There were no significant differences (P≤ 0.05) throughout the period of incubation at concentration I (30 g/L) of all the plant extracts. At concentration II (60 g/L),

  • Period of Incubation (Hours) and Percentage Growth Inhibition (%)
  • Mean
  • 24
  • 48
  • 72
  • 96
  • 120
  • Plant Extract Concentration (g/L)
  • 30
  • 44.44±5.56
  • 51.52±2.69
  • 56.46±2.07 52.47±1.24
  • 54.76±2.38
  • 51.93±1.62
  • 60
  • 100.00±0.00a
  • 66.90±6.56b 65.60±6.49b 58.70±2.44b
  • 61.61±2.80b
  • 70.56±4.35
  • 90
  • 100.00±0.00a
  • 69.93±3.85b 69.10±3.00b 66.21±2.37b
  • 65.51±3.07b
  • 74.15±3.64
  • Piper guineense
  • 30
  • 38.90±20.00
  • 48.48±2.69
  • 54.70±2.20 57.42±1.99
  • 56.76±1.99
  • 51.25±3.93
  • 60
  • 83.30±16.70
  • 64.34±4.90
  • 59.63±1.19 59.98±1.17
  • 60.65±2.20
  • 65.59±3.83
  • 90
  • 100.00±0.00a
  • 67.37±4.82b 64.25±3.21b 66.12±2.88b
  • 64.55±2.72b
  • 72.46±3.88
  • Zingiber officinale
  • 30
  • 50.00±28.90
  • 42.42±7.89
  • 51.91±4.94 46.15±2.22
  • 44.97±2.32
  • 46.95±5.23
  • 60
  • 88.90±11.10a
  • 56.18±6.96b 53.18±0.68b 49.91±2.14b
  • 51.82±2.47b
  • 60.00±4.52
  • 90
  • 100.00±0.00a
  • 56.18±5.36b 57.88±2.75b 57.51±2.23b
  • 59.52±4.83b
  • 66.22±4.72
  • Azadiracta indica
  • 30
  • 38.90±20.00
  • 40.33±6.95
  • 49.53±7.06 43.59±3.39
  • 34.23±2.89
  • 41.31±4.09
  • 60
  • 83.30±16.70a
  • 56.18±5.36b 57.80±3.19ab 51.01±6.34b
  • 43.84±5.01b
  • 58.43±4.83
  • 90
  • 100.00±0.00a
  • 58.74±7.00b
  • 62.49±4.9b 61.08±4.05b
  • 59.52±4.83b
  • 68.37±4.61
  • Carica papaya
  • 30
  • 27.80±14.70
  • 40.79±5.02
  • 43.64±2.83 48.63±3.32
  • 40.12±2.80
  • 40.19±3.32
  • 60
  • 66.70±16.70
  • 56.18±5.36
  • 59.63±1.19 55.04±1.33
  • 49.82±3.15
  • 57.47±3.37
  • 90
  • 100.00±0.00a
  • 62.23±0.69b 64.49±1.10b 61.26±0.27b
  • 62.56±3.15b
  • 70.11±4.05
  • Nicotiana tabacum
  • 4
  • 100.00±0.00
  • 100.00±0.00 100.00±0.00 100.00±0.00
  • 100.00±0.00
  • 100.00±0.00
  • 8
  • 100.00±0.00
  • 100.00±0.00 100.00±0.00 100.00±0.00
  • 100.00±0.00
  • 100.00±0.00
  • 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
  • Mancozeb®

Table 1: Percentage Growth Inhibition of A. ochraceus by different Concentrations of Plant Extracts and Chemical

Means on the same row (for each Plant Extract) with the different superscript are statistically significant Period of Incubation (Hours) and Percentage Growth Inhibition (%) Plant Extract 24 48 72 96 120 Mean I Azadiracta indica 50.00±28.90ab 42.42±7.89b 51.19±4.94bc 46.15±2.22cd 44.97±2.32c 46.95±5.23bc Carica papaya 38.90±20.00b 40.33±6.95b 49.53±7.06bc 43.59±3.39d 34.23±2.89d 41.31±4.09c Nicotiana tabacum 27.80±14.70b 40.79±5.02b 43.64±2.83c 48.63±3.32cd 40.12±2.80cd 40.19±3.32c Piper guineense 44.44±5.56b 51.52±2.69b 56.46±2.07b 52.47±1.24bc 54.76±2.38b 51.93±1.62b Zingiber officinale 38.90±20.00b 48.48±2.69b 54.70±2.20bc 57.42±1.99b 56.76±1.99b 51.25±3.93b Mancozeb 100.00±0.00a 100.00±0.00a 100.00±0.00a 100.00±0.00a 100.00±0.00a 100.00±0.00a LSD 54.58 15.42 12.02 7.22 7.02 9.82 II Azadiracta indica 88.90±11.10ns 56.18±6.96b 53.18±0.68c 49.91±2.14d 51.82±2.47cd 60.00±4.52bc Carica papaya 83.30±16.70ns 56.18±5.36b 57.80±3.19bc 51.01±5.24cd 43.84±5.01d 58.43±4.83c Nicotiana tabacum 66.70±16.70ns 56.18±5.36b 59.63±1.19bc 55.04±1.33bcd 49.82±3.15d 57.47±3.37c Piper guineense 100.00±0.00ns 66.90±6.56b 65.60±6.49b 58.70±2.44bc 61.61±2.80b 70.56±4.35b Zingiber officinale 83.30±16.70ns 64.34±4.90b 59.63±1.19bc 59.98±1.17b 60.65±2.20bc 65.59±3.83bc Mancozeb 100.00±0.00ns 100.00±0.00a 100.00±0.00a 100.00±0.00a 100.00±0.00a 100.00±0.00a LSD 38.91ns 16.55 9.38 8.17 9.23 10.81 III Azadiracta indica 100.00±0.00ns 56.18±5.36c 57.88±2.75c 57.51±2.23c 59.52±4.83b 66.22±4.72b Carica papaya 100.00±0.00ns 58.74±7.00bc 62.49±4.95bc 61.08±4.05bc 59.52±4.83b 68.37±4.61b (P≤0.05) by period of incubation

  • Nicotiana tabacum
  • 100.00±0.00ns
  • 62.23±0.69bc
  • 64.49±1.10bc
  • 61.26±0.27bc
  • 62.56±3.15b
  • 70.11±4.05b
  • Piper guineense
  • 100.00±0.00ns
  • 69.93±3.85b
  • 69.10±3.00b
  • 66.21±2.37b
  • 65.51±3.07b
  • 74.15±3.64b
  • Zingiber officinale
  • 100.00±0.00ns
  • 67.37±4.82bc
  • 64.25±3.21bc
  • 66.12±2.88b
  • 64.55±2.72b
  • 72.46±3.88b
  • Mancozeb
  • 100.00±0.00ns
  • 100.00±0.00a
  • 100.00±0.00a
  • 100.00±0.00a
  • 100.00±0.00a
  • 100.00±0.00a
  • LSD
  • -
  • 13.57
  • 9.12
  • 7.47
  • 10.78
  • 10.78

Table 2: Mean Percentage Growth Inhibition of A. ochraceus at different Concentrations of Plant Extracts and Chemical

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)

1 0 0 P ercenta ge G row th Inh ibition (P lant E xtract)

1 2 0 Tim e of Incubation (H rs) P ip e r g u in e e n s e Z in g ib e r o ffic in a le A z a d ira c ta in d ic a C a ric a p a p a y a N ic o tia n a ta b a c u m • M a n c o z e b Figure 3: Mean percentage growth inhibition of three concentrations of plant extracts (30 g/L, 60 g/L and 90 g/L) and mancozeb (4 g/L, 8 g/L and 12 g/L) on growth of A. ochraceus.

Effect of different concentrations of plant extracts and chemical fungicide in controlling tuber rot of Pepa after five months of storage

Figure 4 shows the effect of different concentrations of plants extracts in controlling rot of Pepa yam tubers after five months of storage. Results revealed that the concentration of 30 g/L of P. guineense on Pepa tuber gave the highest decay reduction index in December, 2015 and February, 2016 with the value of 1each and lowest in March, 2016 and April, 2016 with the value of 0.44 each. Concentration II (60 g/L) recorded the highest value of 1 in December, 2015 and January, 2016 each and the lowest value of 0.66 each for the months of February, March and April, 2016. The performance of the extract at concentration III (90 g/L) was the same throughout the storage period with the decay reduction index value of 1 for each month. The efficacy of Z. officinale at 30 g/L was highest in December, 2015 with the value of 0.66 and lowest in January and February, 2016 with the value of 0.33 for each month. Extract of Z. officinale was most effective at 60 g/L and 90 g/L with the value of 1 throughout the storage period. The effectiveness of A. indica at 30 g/L was similar to 60 g/L with the highest value of 1 in December, January and February each and least in April with the value of 0.66. At 90 g/L the performance rose to the peak of 1 throughout the duration of storage. The synthetic chemical Mancozeb gave an even performance of 1 throughout the period of storage (Figure 4).

Effects of Mean Concentrations of Plant Extract and Chemical Fungicide in Controlling Tuber Rot of Pepa After Five Months of Storage

Table 3 shows the results of the efficacy of mean concentrations of plant extracts (30 g/L, 60 g/L and 90 g/L) and chemical fungicide (4 g/L) in controlling rot of Pepa yam tuber in storage. Results showed that mean decay reduction index in December, 2015 was 1.00 for each of Mancozeb, A. indica and P. guineense while Z. officinale recorded the value of 0.88. In January and February, 2016, Mancozeb and A. indica recorded the mean value of 1.00 each while P. guineense and Z. officinale recorded mean value of 0.88 and 0.77, respectively. Results of March and April were the same for Mancozeb (1.00), P. guineense (0.70) and Z. officinale (0.85) while extract from A. indica recorded the mean value of 0.88 and 0.77 for March and April, respectively. Mean decay reduction index of plant extracts and chemical fungicide after five months of storage of Pepa showed that Mancozeb (1.00) was more effective followed by A. indica (0.93) while P. guineense and Z. officinale recorded similar efficacy of 0.83 each in controlling pathogens of Pepa yam tubers. There was no significant difference (P≤0.05) in potency among the plant extracts for each month of storage of Pepa yam tubers_._ Mean decay reduction index also showed no significant difference (P≤0.05) among treatments (Table 3).

Figure 3: Mean percentage growth inhibition of three concentrations of plant extracts (30 g/L, 60 g/L and 90 g/L) and mancozeb (4 g/L, 8 g/L and 12 g/L) on growth of A. ochraceus.
Click to enlarge
Figure 3: Mean percentage growth inhibition of three concentrations of plant extracts (30 g/L, 60 g/L and 90 g/L) and mancozeb (4 g/L, 8 g/L and 12 g/L) on growth of A. ochraceus.
Plant Extract
Period of StorageLSD
Mancozeb®A. indicaP. guineenseZ. officianale
Ist Year
Dec., 20151.00±0.001.00±0.001.00±0.000.88±0.110.21ns
Jan., 20161.00±0.001.00±0.000.88±0.110.78±0.140.35ns
Feb., 20161.00±0.001.00±0.000.88±0.110.77±0.140.35ns
Mar., 20161.00±0.000.88±0.110.70±0.150.85±0.110.42ns
Apr., 20161.00±0.000.77±0.140.70±0.150.85±0.150.46ns
Mean (1st Year)1.00±0.000.93±0.040.83±0.080.83±0.110.29ns

Table 3: Decay reduction index of some plant extracts and mancozeb after two years of storage on Pepa yam tubers.

2nd Year
Dec., 20160.66±0.330.66±0.160.66±0.160.55±0.170.60ns
Jan., 20170.66±0.330.66±0.160.66±0.160.33±0.160.59ns
Feb., 20170.66±0.330.55±0.170.44±0.170.22±0.140.59ns
Mar., 20171.00±0.000.77±0.140.77±0.140.55±0.170.52ns
Apr., 20171.00±0.000.66±0.160.66±0.160.55±0.170.57ns
Mean (2nd Year)0.80±0.200.66±0.090.64±0.090.44±0.110.36ns
Mean (both Years)
Difference between Years
1st Year1.00±0.000.93±0.04a0.83±0.080.83±0.11a
2nd Year0.80±0.200.66±0.09b0.64±0.090.44±0.11b
LSD0.23ns0.190.26ns0.31

Table 4: Decay reduction index of some plant extracts and mancozeb after two years of storage on Pepa yam tubers.

Means on the same row (comparing plant extracts) and column (comparing year of storage) with different superscript a and b are statistically significant (P≤0.05); ns = not significant

Discussion

Fungi organisms such as F. moniliforme, F. oxysporum B. theobromae, A. flavus, A. niger, A. ochraceus, P. purpurogenum and Pestalotia sp are known to cause rot of yam tubers in storage. This confirms earlier report by Ogaraku and Usman, Ogunleye and Ayansola, Gwa, et al. Okigbo, et al. [5, 8, 22, 35] who isolated different fungi organisms from yam tubers at various locations in Nigeria. Research conducted on Dioscorea sp in Bodija, Ibaban, by Ogunleye and Ayansola [35] revealed that A. ochraceus and Penicillium sp. had low occurrence of 2.14% and 6.43%, respectively. Inoculation of A. ochraceus mycelial into the healthy looking yam tubers produced rot symptoms. The pathogen entered through the holes created in the tubers and probably utilized the nutrients of the tubers as substrates for growth and development during the 14 days of incubation [11, 36]. The tubers that were not inoculated with the test fungus however, did not show any symptom of rot indicating the absent of the rot inciting fungus in the bored yam. Extracts of P. guineense, Z. officinale A. indica, N. tabacum and C. papaya and the synthetic fungicide (mancozeb) all possess fungitoxic compounds that are toxic and capable of inhibiting the growth of A. ochraceus in vitro and controlling rot pathogens in vivo. It has been reported that plant contained secondary compounds such as alkaloids, flavonoids, terpenoids and tannins which have antimicrobial effect against a wide range of fungi [37, 38]. The susceptibility of A. ochraceus to the various plant extracts varied with the duration of incubation, the type of plant extract used as well as the concentrations of the extracts which agreed with earlier report by Gwa and Akombo, Gwa and Nwankiti, Gwa and Ekefan, and Gwa, et al. [9, 10, 19, 22]. Gwa and Akombo and Gwa and Nwankiti [9, 10] evaluated the antifungal activity of A. indica, P. guineense, Z. officinale, C. papaya and N. tabacum against A. flavus and Colletotrichum specie in vitro respectively and they reported that all the extracts significantly reduced the mycelial growth of A. flavus and Colletotrichum species by at least 42% at the different level of concentrations of the plant extracts tested against the pathogens.

P. guineense, Z. officinale were generally more fungitoxic than A. indica, C. papaya and N. tabacum in the in vitro test. This could be as a result of solubility of the active substances in water or higher concentration of the active principles in the seeds and rhizomes of P. guineense, Z. officinale respectively than that of A. indica, C. papaya and N. tabacum. This result is similar with findings of Gwa and Ekefan, Gwa, et al. [19, 22]. Hycenth [39] reported the antifungal effect of A. indica against yam rot pathogens (Rhizopus stolonifer). Contrary to the report of Oluma and Elaigwu [40] who observed that extracts of A. indica had no inhibitory effect on the mycelial growth and sclerotial formation of Macrophomina phaseolina. It has been shown that A. indica contains phytochemical compounds such as azadirachtin, betasiterol, 6-desacetyl nimbinene and 3- desciacetyl alamine which show antifungal properties. The growth inhibition of A. ochraceus by P. guineense extracts may be probably due to the presence of phyto-chemical compounds such as piperine which increases as the concentration of P. guineense increased against A. ochraceus. Ijato [41] studied the antifungal effects of Allium sativum (rhizome) and Nicotiana tobacum (leaf) extracts on rot causing organisms on yam against Aspergillus niger, Fusarium oxysporum, Rhizopus stolonifer, Botryodiplodia

theobromae, Aspergillus flavus and Fusarium solani and found out that both the aqueous and the ethanolic extract of the tested plants were effective as bio-killer on yam rot organisms. According to Suresh, et al. and Wang, et al. [42, 43] the inhibition of pathogens increased as the active compound (nicotine) in N. tabacum increased. Taiga, et al. [44] showed that N. tabacum cold extract inhibited the mycelia of F. oxysporum yam rot organism. The inhibition of A. ochraceus by Z. officinale may be attributed to the presence of an active ingredient called gingerol which acted against the growth of A. ochraceus in culture. Chohan and Perveen [45] inhibited the growth and spore germination of F. oxysporum and F. solani, causal agents of wilt and root rot, respective of tomato using Curcuma longa Val., Allium sativum L. and Zingiber officinale Rosc. Okigbo and Nneka [26] suppressed the growth of rot fungi in culture and reduce rot development in yam tubers using Z. officinale. The active compound papain in C. papaya may be responsible for its inhibitory activity [46]. Suleiman [47] inhibited mycelial growth of Alternaria solani, causal organism of yam rot using leaf extracts of C. papaya while Chima [48] showed that leaf extracts of C. papaya contained phytochemical such as tannins, glycosides, alkaloids, and flavonoids that are responsible in controlling post harvest soft rot of yam caused by Rhizopus nigricans and Mucor circinelloides.

Effect of concentrations of P. guineense, Z. officinale, A.indica and chemical fungicide in controlling rot pathogens of Pepa tubers in storage for five months revealed that P. guineense, Z. officinale and A. indica extracts possess antimicrobial activities against rot pathogens of yam in storage at different concentrations. The results revealed that 60 g/L and 90 g/L were more effective than 30 g/L of the extracts. The differences in the activities of the extracts may be due to the presence of secondary metabolites in these plants. Mean decay reduction index (DRI)for each of the plant extract and chemical fungicide on Pepa cultivar of white yam tubers tested showed mean values from 0.83 (P. guineense and Z. officinale), 0.93 (A. indica) to 1.00 (mancozeb). This shows that the plant extracts reduced the occurrence of yam tuber rot disease by between 83% and 100% during storage of yam tubers for five months. This showed that only 17% of tubers treated with P. guineense and Z. officinale and 7% of tubers treated with A. indica extracts were lost to storage pathogens in the first year. The second year showed mean decay reduction index between 0.44 (Z. officinale) and 0.66 (A. indica) and 0.80 (mancozeb). This showed that the extracts controlled between 44% and 66% of rot pathogens and 80% of rot pathogens when mancozeb was used. This result disagreed with investigation obtained by Okigbo, et al. [11] who recorded high rot reduction of 62.80% using A. sativum to control pathogens of yam_. Similarly, Udo, et al. [49] reduced the growth and sporulation of fungal pathogens on sweet potato and yam with garlic (_A. sativum). The result also disagreed with findings of Aidoo [50] who used mancozeb to control yam tuber rot diseases of dente and pona and recorded decay reduction index of 0.46 and 0.60 respectively which implies that only 46% and 60% of rot pathogens were controlled in dente and pona respectively contrary to the 100% and 80% inhibition achieved in the first and second year, respectively.

Conclusion

The study demonstrated the potency of A. indica, P. guineense, Z. officinale, C. papaya and N. tabacum extracts against A. ochraceus in vitro and A. indica, P. guineense and Z. officinale against other yam rot pathogens in storage. The effectiveness of A. indica was more compared with P. guineense and Z. officinale on storage Pepa yam for five months both in the first and second year of storage. All the other extracts are also found to be effective and could be exploited for the management of A. ochraceus and other yam pathogens in storage since they are eco- friendly. The cheapness and availability of these plants will also make it possible for the development of natural plant protection products at the disposal of peasant farmers.

Conflict of Interest Disclosure

The authors declare that there is no conflict of interest regarding the publication of this paper.

Funding Acknowledgement

This research received no specific grant from any funding agency in the public, commercial or not-for- profit sectors.

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@article{gwa2018,
  title   = {Antimicrobial Activity of Five Plant Extracts and Synthetic
Fungicide in the Management of Postharvest Pathogens of Yam
(Dioscorea Rotundata Poir) in Storage},
  author  = {Gwa VI, Nwankiti AO and Hamzat OTH},
  journal = {Open Access Journal of Microbiology & Biotechnology},
  year    = {2018},
  volume  = {3},
  number  = {1},
  doi     = {10.23880/oajmb-16000128}
}
Gwa VI, Nwankiti AO and Hamzat OTH (2018). Antimicrobial Activity of Five Plant Extracts and Synthetic
Fungicide in the Management of Postharvest Pathogens of Yam
(Dioscorea Rotundata Poir) in Storage. Open Access Journal of Microbiology & Biotechnology, 3(1). https://doi.org/10.23880/oajmb-16000128
TY  - JOUR
TI  - Antimicrobial Activity of Five Plant Extracts and Synthetic
Fungicide in the Management of Postharvest Pathogens of Yam
(Dioscorea Rotundata Poir) in Storage
AU  - Gwa VI, Nwankiti AO and Hamzat OTH
JO  - Open Access Journal of Microbiology & Biotechnology
PY  - 2018
VL  - 3
IS  - 1
DO  - 10.23880/oajmb-16000128
ER  -