Journal of Plant Pathology & Microbiology

Yam (Dioscorea rotundata Poir) is an important food crop in West Africa, the Caribean’s, South America, and South-East Asia [1] Production and consumption of yam is mostly in West Africa with Nigeria been the highest producer with about 38.92 million metric tonnes annually [2]. The production and storage of yam is constrained by several factors such as pathogens mostly from fungi which are considered heavy in Nigeria [3-5]. Rot of yam tubers in storage caused by pathogenic organisms is of particular importance because the pathogens reduce viability of the tubers and subsequently yield and quality [6,7]. Fungi implicated as rot causing organisms of yam tubers in storage include Fusarium oxysporum, F. moniliforme, F. solani, Penicillium chrysogenum, P. oxalicum, P. purpurogenum, P. digitatum, Rhizopus nodosus, Rhizoctonia spp, Aspergillus flavus, A. ochraceus, A. niger, Colletotrichum spp, Botryodiplodia theobromae, [5-13]. Fungi pathogens have also been reported to be major cause of storage rots of cassava and sweet potatoes [14-16]. Synthetic chemicals have been used in reducing rot of yam tubers both in vitro and in vivo [3,17-20]. These chemical pesticides are found to be costly, may even cause environmental pollution and induce pathogen resistance [21,22]. Pesticides of plant origin are generally considered to be better in controlling pathogens of plants both in the field and in storage barns because they are cheap, biodegradable, safe, easily available and environmentally free from pollution compared with the chemically formulated fungicides [23,24]. Ability of plants to synthesize aromatic secondary metabolites such as coumarins, flavones, flavonoids, flavonols phenols, phenolic acids, quinines, tannins, terpenoids, alkaloids [25,26] have made them effective against rot-causing pathogens [26-28]. Thousands of these phyto-chemicals metabolites have been demonstrated to have inhibitory effects on all types of pathogens in vitro and are therefore subjected to in vivo testing in order to evaluate the efficacy of the plant extracts in controlling diseases in crops [19,27,29,30]. It is against this backdrop that different plants were selected and formulated to test their potencies in inhibiting pathogens of yam in storage hence could be considered as alternatives in the control of pathogens of yam both in culture and in the field.

Experimental site

The experiment was carried out at the Advanced Plant Pathology Laboratory, Federal University of Agriculture, Makurdi, Nigeria.

Collection of diseased yam tubers

Diseased white yam (Dioscorea rotundata) varieties (Ghini, Hembankwase) showing different types of symptoms were collected from farmers’ barns in Kadarko, Keana local government area of Nasarawa State, Nigeria. The settlement is located between longitudes 8°30’ and 8°35’E, and latitudes 8°10ʹ and 8°14′N. Tubers were collected and sealed in sterile polyethylene bags to prevent wounding and possible infection by other pathogenic organisms. The tubers were taken to the laboratory for subsequent isolation and identification of rot causing fungi two days after collection of samples. Potato Dextrose Agar (PDA) medium was prepared according to manufacturer’s recommendation and used for isolation of the rot causing fungi pathogens. Pathogenicity tests were done using the healthy yam tubers collected from same location.

Isolation and identification of rot causing fungi

Diseased yam tubers showing different symptoms of rots were washed in clean water before cutting into small pieces of about 2 × 2 mm from the advancing edge of lesion using sterile scalpel. A 5% Sodium hypochlorite solution was used to surface sterilized the cut tissues for 2 minutes in order to clean them off from contaminants. The sterilized cut pieces were then rinsed in four successive changes of sterile distilled water in order to remove the chemical before drying on sterile filter paper for 5 minutes [31]. Plates containing sterile solidified potato dextrose agar (PDA) were inoculated with four pieces of the sterilized diseased yam. Inoculated plates were neatly covered with mastic tapes to prevent contamination by air borne fungi and incubated at ambient room temperature (30 ± 5°C) for 7 days. The growths of the fungi were observed daily. Fungi colonies were sub-cultured and identified after 7 days of incubation when pure cultures were fully grown [31]. Different fungal colonies were seen from the rotted yam samples, from which B. theobromae, A. flavus, A. niger, A. ochraceus, F. moniliforme, F. oxysporum, F. solani, C. eragrostide and Colletotrichum sp were identified, purified and multiplied on PDA. F. solani wass choosen for further studies since it has not been previously isolated in this location. Identifications of grown pure cultures the fungi were done using morphological characteristics as well as microscopic and identification guide [32,33].

Pathogenicity tests

Pathogenicity tests of B. theobromae, A. flavus, A. niger, A. ochraceus, F. moniliforme, F. oxysporum, F. solani, C. eragrostide and Colletotrichum sp isolated from the two varieties of rotted yam lesions were confirmed by inoculating these organisms into healthy Hembankwase yam tubers. Running tap water was used to wash the healthy-looking yam tubers before sterilization with a 5% Sodium hypochlorite solution for 2 minutes. Tubers were then rinsed in four successive changes of sterile distilled water. Cylindrical discs of 5 mm were removed from the healthy yam tubers using a sterile cork borer. Mycelial discs of each fungus measuring 4 mm in diameter were taken from 5 day-old cultures of each of the fungi and each fungal disc was put into the hole created in the tubers. The control experiments had sterile agar discs inserted in the holes created instead of the inoculum which was obtained from the fungi [19]. Sterile petroleum jelly was used to completely seal the remaining parts of the holes to prevent pathogenic invasion. The inoculated yam tubers were replicated three times for each of the tested pathogens and control experiments and were arranged in completely randomized design. The treatments were incubated at ambient room temperature (30 ± 5°C) under sterile condition for 14 days for growth of the fungi to establish. When growths were fully established, the 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 the pathogens after the incubation period were compared with those naturally infected tubers initially collected from farmers’ barns. Re-isolation of the fungi pathogens from the inoculated diseased yam tubers were done and cultured on PDA plates. The characteristic culture of each of the pathogens obtained was compared with the culture initially collected from the naturally infected tubers.

Preparation of plant extracts

The preparations of plant extracts were carried out using the methods of Gwa and Akombo, [11] and Gwa and Nwankiti [12]. Extracts prepared from healthy plant materials were from leaves of Azadirachta indica (Neem), rhizomes of Zingiber officinale (Ginger), seeds of Piper guineense (Black Pepper), leaves of Nicotiana tabacum (Tobacco) and leaves of Carica papaya. These materials were washed neatly with cold running tap water; air-dried and separately ground into fine powder using pestle and mortar. About 30 g, 60 g and 90 g of the ground plant materials were dissolved in 1000 ml of sterile distilled hot water (100°C) separately in 1500 ml Pyrex flask. The mixtures were stirred and left for 24 hours and subsequently filtered using four-fold of sterile cheese cloth. These gave concentrations of 30 g/L, 60 g/L and 90 g/L, respectively. The filtrates obtained were used as the plant extracts in the experiment. Mancozeb, a chemical fungicide was prepared by dissolving 4 g, 8 g and 12 g in one litre of sterile distilled water separately to give concentrations of 4 g/L, 8 g/L and 12 g/L, respectively. The potencies of the extracts and the chemical fungicide were compared for their in vitro fungicidal activity in inhibiting mycelial growth of F. solani.

Effect of plant extracts on F. solani

The effects of the prepared extracts were evaluated on radial mycelial growth of F. solani using the method of Amadioha and Obi [34]. Four equal sections were created at the bottom of the plates by drawing two perpendicular lines. The intersections of the two lines were considered as the centre of the plates before dispensing PDA into each of the plates. About 15 ml of the prepared medium was poured into sterilized Petri dishes containing 5 ml of each plant extract and chemical fungicide at their respective concentrations [35], 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 5 mm diameter of one-week-old cultures [36] of F. solani. The treatments were replicated three times for each of the concentrations. In the control experiments, 5 ml of sterile distilled water was added to PDA in place of plant extracts respectively; the treatments and control were completely randomized [37] and incubated for 120 hours at ambient room temperature (30 ± 5°C).

Measurement of mycelial radial growth of F. solani

Growth of F. solani was measured after 24 hours for five consecutive times using a transparent ruler. The absence of growth in any of the plates was an indication of the efficacy of the extracts and the chemical fungicide against F. solani. Fungitoxicity was calculated as percent growth inhibition (PGI) of F. solani over control based on the formula stated by Korsten and De Jager [38].

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 potencies of the aqueous plant extracts and the chemical fungicide that were found to be effective against F. solani in vitro were used to control other seed pathogens of yam in the field.

Potency of plant extracts and chemical fungicide in controlling rot causing pathogens of white yam setts planted in the field

The potencies of the five plant extracts in controlling yam tuber rot in vitro were tested on yam setts planted in vivo. The extracts were applied on four cultivars of white yam namely: Ogoja, Pepa, Hembankwase and Ghini to test their efficacies in controlling rots pathogens on germination of white yam setts in the field using the different aqueous extract concentrations. The four cultivars were each sprayed with the five botanical extracts at concentration of 30 g/L, 60 g/L and 90 g/L, respectively while mancozeb was applied at concentrations of 4 g/L, 8 g/L and 12 g/L, respectively using a hand sprayer. After spraying, the tubers were allowed to dry before planting. The treatments were completely randomized, and control treatments were sprayed with sterile distilled water (no plant extract or chemical applied). The experiments were monitored regularly for the first eight weeks after planting to allow enough time for germination of all the tubers. The tubers that did not germinate were removed thereafter and examined for signs of rots. The numbers of unrotten (germinated) and rotten (ungerminated) tubers in each treatment were recorded for Ogoja, Pepa, Hembankwase and Ghini cultivars, respectively. The effectiveness of each plant extract and chemical fungicide in controlling yam tuber rot and increasing germination of yam setts at different concentrations was calculated using the Decay Reduction Index (DRI) as proposed by Amadioha [39].

Statistical analysis

Analysis of variance (ANOVA) using GenStat Discovery Edition 12 for ANOVA and means separation and Graph Pad Prism 6 for trend graphs. Statistical F-tests were evaluated at p ≤ 0.05 using Fisher’s least significant differences (FLSD) [40].

Isolation of fungal pathogens from rotted yam tubers

B. theobromae, A. flavus, A. niger, A. ochraceus, F. moniliforme, F. oxysporum, F. solani, C. eragrostide and Colletotrichum sp., were isolated from Hembankwase and Ghini cultivars of white yam tuber samples collected from farmers’ barns in Kadarko a major yam producing settlements in Nasarawa State, Nigeria. Figure 1 shows the characteristics pure culture of F. solani which was chosen for this study because it has not been previously studied in this location. The fungus was grown on Potato dextrous agar (PDA) as shown in Figure 2.

Figure 1: Culture of F. solani on Potato Dextrose Agar (10x).

Figure 2: Photomicrograph of F. solani showing macroconidia (10x).

Pathogenicity tests

Pathogenicity tests conducted on B. theobromae, A. flavus, A. niger, A. ochraceus, F. moniliforme, F. oxysporum, F. solani, C. eragrostide and Colletotrichum sp using Hembankwase cultivar of yam tubers show that all the organisms were pathogenic. Results presented in Figure 3 show that F. solani induced rot in healthy looking Hembankwase cultivar of yam 14 days after inoculation. Re-isolation of the test organism, F. solani from the artificially inoculated yam tubers shows symptoms similar with the naturally infected tubers. Tubers that were not inoculated with F. solani mycelial (control) did not show symptoms of rot in the hole created in the yam tubers (Figure 4).

Figure 3: Rot caused by F. solani.

Figure 4: Control (without F. solani inoculum).

Effect of some plant extracts on growth of F. solani in vitro

Results presented in Table 1 show that P. guineense, Z. officinale, A. indica, C. papaya and N. tabacum extracts possess fungicidal properties at different concentration levels against mycelial growth of F. solani in vitro. However, only mancozeb gave 100% inhibition of mycelial growth of F. solani. All the plant extracts were able to reduce the radial growth of the test fungus throughout the period of incubation irrespective of the concentration level used. 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, Z. officinale and A. indica were considered better extracts compared with C. papaya at all the levels of concentrations. At concentration I (plant extract 30 g/L and mancozeb 4 g/L), the percentage growth inhibition of F. solani decreased at 24 hours from 79.44%, 79.44%, 73.89%, 75.60%, 53.33% and 100% to 48.20%, 48.27%, 35.54%, 29.75%, 23.92% and 100% at 120 hours with P. guineense, Z. officinale, A. indica, C. papaya, N. tabacum and mancozeb, respectively. At concentration II (plant extract 60 g/L and mancozeb 8 g/L), the percentage growth inhibition of F. solani decreased at 24 hours from 100%, 87.89%, 79.44%, 93.33%, 60.00% and 100% to 55.22%, 55.18%, 47.04%, 39.11%, 34.39% and 100% after 120 hours with P. guineense Z. officinale, A. indica, C. papaya, N. tabacum and mancozeb, respectively. At concentration III (plant extract 90 g/L and mancozeb 12 g/L), the percentage growth inhibition of F. solani decreased at 24 hours from 100%, 93.33%, 79.44%, 94.44%, 80.60% and 100% to 61.96%, 63.11%, 56.32%, 56.09%, 43.51% and 100% at 120 hours in P. guineense, Z. officinale, A. indica, C. papaya, N. tabacum and mancozeb respectively (Table 1). There were significant differences (p ≤ 0.05) in the activity of plant extracts at each level of concentration throughout the period of incubation (Table 2). Mean percentage growth inhibition of F. solani after 120 hours of incubation revealed increase in the performance of the extracts from the lowest concentration to the highest concentration. The most potent extracts at concentrations I (30 g/L) were P. guineense (57.94%), followed by Z. officinale (57.69%), A. indica (52.71%), C. papaya (42.48%) and N. tabacum (28.25%) respectively while mancozeb consistently gave 100% inhibition in F. solani throughout the period of incubation and at all level of concentrations respectively (Table 2). Mean percentage growth inhibition of three concentrations (I, II and III) of each plant extract and chemical fungicide showed that P. guineense and Z. officinale were the most effective extracts in reducing the mycelial growth of F. solani in culture throughout the period of incubation (Figure 5).

Table 1: Percentage growth inhibition of F. solani at concentrations of plant extracts and chemical fungicide after 120 hours of incubation in culture.

Table 2: Comparative assessment of plant extracts and chemical fungicide at different concentrations after 120 hours of incubation.

Figure 5: Mean of three concentrations of each plant extract (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 F. solani.

Effect of plant extracts and chemical fungicide in controlling rot causing pathogens of white yam setts planted in the field

Table 3 compares the potency of some plant extracts and chemical fungicide in controlling rots causing pathogens of white yam setts planted in the field using decay reduction index method. Result showed that all the plant extracts were able to inhibit growth of rot causing pathogens on all the cultivars of white yam setts planted for 2015 and 2016 seasons. In 2015, P. guineense was more potent on Pepa (0.77) cultivar followed by Ogoja (0.66) while the least inhibition was recorded on Hembankwase (0.33) cultivar. Z. officinale was more effective on Pepa (0.88) but least on Hembankwase (0.22). A. indica (0.88) N. tabacum (0.66) and Mancozeb (0.77) respectively gave a higher decay reduction index value in Pepa compared with Hembankwase which recorded values of 0.33 for each of the extracts respectively. C. papaya was however, more potent on Ogoja (0.66) compared with Hembankwase (0.33). Only Z. officinale and A. indica gave significant difference (p ≤ 0.05) among the four cultivars of white yam setts tested in the field. There was also no significant difference (p ≤ 0.05) among extracts for each cultivar of white yam tested. The result of 2016 revealed that only P. guineense proved more effective on Pepa (0.77) cultivar but less effective (0.66) on other cultivars respectively. There was no significant difference (p ≤ 0.05) among cultivars for each plant extract tested and no significant difference among extracts for each cultivar used. Mean decay reduction index for two years for each of the cultivars used and each of the extracts tested showed no significant differences for each extract and for each cultivar respectively (Table 3).

Table 3: Mean decay reduction index of some plant extracts and chemical fungicide against rot-causing pathogens of white yam setts in the field.

Chemical fungicides have been found to be most effective in the control of fungal pathogens of crops both in the field and in store. In order to find out fungicides which are effective, plant fungicides were tested at different concentrations both in vitro and in vivo. The result revealed that P. guineense, Z. officinale, A. indica, C. papaya and N. tabacum and the synthetic chemical; mancozeb all possess’ antimicrobial properties potent enough to inhibit mycelial growth of F. solani in culture. The inhibitory activity of F. solani by the different plant extracts depend on the magnitude of concentration as well as the duration of incubation and the type of plant extract used [11,17,41]. Percentage growth inhibition of F. solani increased with increase in concentrations of plant extracts indicating that the effects of the active compounds of the extracts used were persistent and increased with the incubation period [11,12,42,43]. This result is similar to the finding of [19] that P. guineense, Z. officinale, A. indica, C. papaya and N. tabacum significantly inhibited the growth of B. theobromae in culture at higher concentrations compared to lower concentrations. The inhibition of F. solani by P. guineense extracts may be attributed to presence of phyto-chemical compounds such as piperine. Antimicrobial activity of piperine increased as the concentration increases against F. solani. Similar study was carried out by Aidoo, [44] who used the seed extract of P. guineense and rhizome of Z. officinale to reduce the growth of B. theobromae and F. oxysporum on two varieties of white yam (D. rotundata and D. alata).

The study also revealed that the rhizome extract of Z. officinale completely inhibited the growth of F. solani at different concentrations. Result of Okigbo and Nneka [45] and Gwa and Akombo [11] confirmed that Z. officinale inhibited the growth of rot fungi in culture and reduces rot development in yam tubers. Results obtained by Anukwuorji et al., [46] revealed the abundance of saponins in Z. officinale was responsible for inhibition of pathogens.

In other studies, conducted by Suresh et al. [47] Wang et al. [48] and Gwa et al. [19] the higher the concentration (nicotine) of N. tabacum the more the inhibition of mycelial growth of pathogens. This is in agreement with the result obtained here as the growth of F. solani was reduced more at higher concentrations compared with application of N. tabacum at lower concentration. Amadioha and Obi [34] showed that A. indica (neem) and Xylopia aethiopica seed extracts have fungitoxic activity against the anthraconose fungus (Colletotrichum lindemuthianum) of cowpea. The inhibitory activity of C. papaya is attributed to its active compound papain [26,49] C. papaya, C. odorata and Acalypha ciliata has proved very effective against pawpaw fruit rot fungi. In similar studies, Ogwulumba et al. [50] used C. papaya leaf extracts to reduce incidence of foliar mycopathogens of groundnut (Arachis hypogea) while result obtained by Suleiman [51] showed the inhibition of mycelial growth of Alternaria solani, causal agent of yam rot using leaf extracts of C papaya. The inhibitory activity of C. papaya may be due to the presence of phyto-chemical compounds such as tannins, glycosides, alkaloids, and flavonoids [52].

Study has shown that A. indica as a medicinal plant possesses potent antifungal properties in the leaves which inhibit the growth of pathogens both in-vitro and in-vivo. According to Amadioha [53], A. indica contains phytochemical compounds such as azadirachtin, betasiterol and 3- desciacetyl alamine which show antifungal properties. Hycenth [54] demonstrated the antimicrobial effect of A. indica against yam rot pathogens (Rhizopus stolonifer). Mallesh and Narendrappa [55] showed that leaf extract of A. indica caused maximum inhibition of mycelial growth of F. solani and R. solani. Similarly, Yelmame et al., [56] showed inhibitory effect against F. solani using neem cake. Mohammad et al., [57] investigated antifungal activity of ten plant extracts under laboratory conditions and they found out that only higher doses of A. indica and Calotropis procera extracts caused maximum inhibition followed by Citrus hystrix and Capsicum annuum extracts. The five extracts (P. guineense, Z. officinale, A. indica, C. papaya and N. tabacum) and chemical fungicides (Mancozeb) which were applied on yam setts before planting were able to protect the yam setts against rot pathogens by 22% in Hembankwase and 77% in Pepa varieties. It was observed that the second year of planting gave a similar but better result with all the plant extracts and the chemical fungicide reducing rot by at least 66% in each cultivar of the white yam setts. The reason for the occurrence of more disease in the first year could probably be due to the interaction between the pathogens, host and environmental conditions which favoured the production of inoculum in the first year more than in the second year hence a better control in the second year. This result agreed with the work of Ekundayo [58] and Agrios [59] who found that favourable environmental condition favoured the production of inoculum. The work is similar to the result obtained by Aidoo [44] who recorded the decay reduction index value of Mancozeb in controlling the rot of ‘dente’ white yam tuber at 0.46 and that of ‘pona’ white yam tuber at 0.60 which corresponded to reduction in rot by 46% for ‘dente’ and 60% for ‘pona’ varieties of white yam, respectively. It was observed that Z. officinale and A. indica extracts significantly differed across varieties in the first year and that all the extracts were more potent on Pepa cultivar but less effective on Hembankwase cultivar in both years. The different levels of reductions of rot by the plant fungicides may probably be due to the varying extent of interference of the different phytochemical compounds with the metabolism of the rot organisms involved.

Yam tubers are susceptible to different fungal pathogens both in storage and in the field. Crude extracts of plant from P. guineense, Z. officinale, A. indica, C. papaya and N. tabacum and the synthethetic chemical mancozeb could be used in controlling pathogens of yam both in storage and in field. Since chemical fungicides are costly, non-biodegradable, toxic to man and environment, it is therefore, recommended that application of plant extracts as natural fungicides for the control of different plant pathogens of yam setts before planting be adopted for small-scale farmers to reduce rot of yam setts and increase germination as well as increase shelf life of harvested produce.