Yam (Dioscorea Spp) belongs to the family Dioscoreaceae. It is a herbaceous annual climbing plant with edible underground tubers¹. Yams have been reported to comprise of about 600 species worldwide^2,3. Ngo-Ngwe et al.⁴ reported that yam is considered a famine food and plays a prime role in the food habits of small and marginal rural families and forest-dwelling communities during the food scarcity periods. Viruel et al.⁵ pointed out that it is recognized as the fourth most important tuber crop after cocoyam, cassava, and sweet potatoes and contributes about 10% of the total root and tubers production around the world.

White yam (Dioscorea rotundata Poir.) according to Onwueme^6,7 is the most important species of yam in West Africa. It is a monocotyledonous, perennial herb cultivated for the consumption of its starchy tuber⁸. Okigbo et al.⁷ reported that yam tuber is chiefly the main useful part of the crop. Its prepared in diverse ways before consumption, it maybe pounded,  roasted, boiled or fried fried. Opara and Nwokocha⁹ reported that yam is essential in food security especially in the tropics this is because it has a relatively longer shelf life than most crops.

Yam rot according to Okigbo et al.¹⁰ is the biological degradation of yam tubers in storage facilities or in yam barns. It reduces both the quality and quantity of yam tubers whether in storage or before consumption. Amusa et al.¹¹, Anukwuorji et al.¹² reported that yam rots or deterioration of cultivated yam usually starts in the soil and progresse to other stages of growth and processing within the value chain. Tuber rot or spoilage occurs even when there is no any external sign of symptom or infection in the infected tuber. Yam spoilage is common in an environment with very high moisture content in the atmosphere; this is because high humidity and temperature of 25-39^oC encourage the growth and proliferation of the rot inducing microorganisms¹³.

Biofungicides according to Dicklow¹⁴ are formulations of living organisms that are used to control the activity of plant pathogenic fungi and bacteria. Biofungicides are microorganisms that are applied to the soil or foliage to prevent or control pathogenic bacterial or fungal infections in plants and plant roots. Diclow¹⁴ reported that these microorganisms produce a wide range of antibiotic substances, parasitize and compete with other fungi, and induce localized or systemic resistance in plants. Antagonistic microorganisms according to Mokhtar and Aid¹⁵; Gwa et al.¹⁶ can compete with the pathogen for food, stops the growth and proliferation of pathogenic organisms either by mycoparasitism or by by secreting antibiotics or toxins.

Biological control method using Biofungicides has several advantages when compared to chemical products and are far more economical than plant extracts. Okigbo¹⁷ reported that biological control of plant disease is has beneficial effects over chemical pesticides. Anuagasi et al.¹⁸ reported that biocontrol agents are ecofriendly, mild on non-target organisms and decompose easily in the environment. Furthermore, Okigbo and Ikediugwu¹⁹ reported instances where biological control has showed to last longer than chemical control substances in its effects and in most cases does not require regular applications.

This research is aimed at evaluating the antagonistic potential of some Biofungicides on storage rot-inducing fungi.

MATERIALS AND METHODS

Preparation of Culture Media

Potato Dextrose Agar (PDA) and Nutrient agar (NA) were used as the media for the growth and maintenance of the fungal and bacterial isolates. The PDA and NA were prepared in accordance with the manufacturer’s instructions. The prepared media was transferred to a Pyrex media bottle and sterilized in the Autoclave at 121^oC, the pressure of 15 (Psi) for 15 minutes²⁰. After sterilization, the medium was allowed to cool to about 45^oC before aseptically dispensing into 15ml aliquots to sterile glass Petri dishes which were allowed to cool down and gel. After solidification, each agar plate was aseptically wrapped round with masking tape until needed for use. These plates were stored in the refrigerator.

Isolation of Fungal Pathogens from Rotted Yam Tubers

The method was described by Ritichie²¹, and modified by Okigbo et al.⁷. Yam tubers with symptoms of rot were surface sterilized by immersing totally in a 10% concentration of sodium hypochlorite solution for 2 minutes. This step was to remove surface contaminants. Rotten yam tubers were washed and rinsed in sterile distilled water and surface sterilized again by cleaning with 70% ethanol-soaked cotton wool. The yam tubers were cut open with a surface sterilized kitchen knife to reveal the boundary area between the healthy tubers and the rotten side. Innocula were taken as pieces cut off with flamed scalpel (5mm by 5mm) in size. The yam pieces were then placed on sterile paper towels in the Laminar Air Flow Cabinet to dry for 5 minutes. Some of the Innocula were transferred directly into sterile PDA plates, while others were first teased out in sterile distilled water. Innocula or loopfuls of the resulting suspension was transferred into sterile PDA plates and spread evenly over the surface. All the plates were incubated “up side down’ in an incubator at room temperature (28 to 32^oC) for 2 to 5 days. The plates were however observed daily for fungal growth and development. When growth has established, sub-cultures were prepared using Innocula from the different organisms in the mixed culture as obtained.

Sub-culturing and Purification of the Fungal Isolates Pathogenic to Yam Tubers

The test fungi were purified by transferring hyphal tips from the colony edges to fresh plates of PDA with the aid of flame-sterilized blades²⁰. These plates were incubated in the incubator at 28 to 32^oC for 2 to 5 days and observed daily for fungal growth and development. Several subculturing was done thus, purifying the mixed culture plates. Stock cultures were prepared by using the surface sterilized fungi which were consigned into McCartney slant bottles of acidified PDA and stored in a refrigerator for characterization and further studies^{7, 22}.

Identification and Characterization of Fungal Isolates

Identification of fungal isolates was based on the morphological or structural features as seen on the culture plates as well as slides viewed under the compound microscope. Microscopic examination involved slide mounts of test isolates stained with Lactophenol Cotton Blue Stain. Morphological or structural features as observed from each isolate were matched against those present in standard manual or identification guides²³.

Determination of Percentage Frequency of Occurrence of the Fungal Isolates

This was done to determine the incidence of occurrence of the different fungal isolates associated with yam tubers. The total number of each isolate was obtained against the total number of all the isolates in all the samples screened. Frequencies of occurrence of different fungal isolates were therefore determined using methods described by ^{24, 10}. Number of times each fungus was encountered was recorded and the percentage frequency of occurrence was calculated using the formula described by Ebele²⁵ thus:

Number of times a fungus was encountered × 100   

                                    Total fungal isolations

Pathogenicity Test

A pathogenicity test was carried out to ascertain that the fungal isolates from rotted yam samples were able to induce rot or otherwise on healthy yam tubers. The methods of Okigbo and Odurukwe²⁶; Okigbo and Emeka¹³; Anukwuorji et al.¹² were adopted. Healthy yam tubers were washed with tap water to remove depris or soil. According to Ritichie²¹ the yam tubers were surface sterilized to expunge surface contaminats by immersing the tubers in 10% concentration of Sodium hypochlorite for 2 minutes and rinsing twice in sterile distilled water. To further remove contaminants, the yam tubers were surface sterilized with 70% ethanol solution. The tubers were then placed on sterile paper towels in the Laminar Air Flow Cabinet to dry for 20 minutes. A sterile cork borer (5mm diameter) was used to bore cones (holes) into the yam tubers at marked points. The cylindrical fleshy portions that were removed from the tubers at each point were kept in sterile Petri dishes. Innocula from the growing culture of each test isolate was introduced into the hole made and the reserved cylindrical part was carefully replaced after inoculation. Part of the tuber removed earlier was cut off to make up for the thickness of the agar inoculum. The replaced core and edges at the point of inoculation was tightly sealed with petroleum jelly to prevent contamination and labelled accordingly. A control experiment was set up by inoculating another tuber with 1ml of sterile distilled water without the fungal isolates. After inoculation, all the tubers were incubated for 14 days at a temperature of 28 to 30^oC and examined daily for evidence of rot such as discoloration, softening, offensive odour, etc. After 14 days, the tubers were carefully cut open along the line of inoculation to reveal the main portions of the tubers which were then examined for the extent of rot. Where positive, the length and girth of the rotted area and those of the entire tubers as revealed were measured and recorded.

Isolation, Purification and Identification of Bacterial Antagonist (Biofungicides)

Isolates of Bacillus subtilis and Pseudomonas syringae with accession numbers, KM972670 and KT887197.1. respectively were obtained using the methods of Okigbo (2002); Okigbo and Emeka (2010) from the topsoil of a farm at NRCRI, Umudike, and from naturally infected tomato fruits showing typical bacterial speck disease symptoms collected from an open market in Umuahia. A ten fold serial dilution (w/v) of the soil in sterile distilled water was first heated to 100^oC for 10 min in a water bath. A sample (0.1 ml) of 10³ dilutions in the water of the soil was spread on yam dextrose agar (YDA) and incubated at room temperature (28 to 30^oC) for up to 48 hour during which characteristic colonies of Bacillus developed. Isolates were subjected to preliminary microbiological analysis and were further identified using molecular characterization. The tomato fruits were rinsed thrice with sterile distilled water and uniformly segmented. The dissected tomato fruits were surface sterilized with sodium hypochlorite (2% solution) for 3 to 5 minutes followed by rinsing with sterilized distilled water thrice. Subsequently, the tissues were pulverized in 2 ml potassium phosphate buffer (0.05 M) followed by incubation at room temperature for 10 min. Afterwards, the homogenate was streaked on nutrient agar (NA) medium plates with a sterilized inoculating loop (Cheesbrough, 2004). Plates were incubated at 27^oC for 48 hours and the germinated bacterial colonies were observed. Bacterial colonies were purified by transferring a single colony culture to a new nutrient agar medium plate to gain the pure bacterial culture that was maintained at 4^oC for in glass slants containing NA medium for further studies.

Isolation, Purification and Identification of Fungal Antagonist (Biofungicides)

Trichoderma harzianum and Trichoderma viride with accession numbers OR227920.1 and ON208727 respectively were isolated from the surface of spoilt tomato fruits collected from an open market in Umuahia town using the method of Okigbo and Emeka (2010). Each was placed in a 500ml beaker containing 200ml sterilized distilled water placed on a rotary shaker at 100rpm for 12 hours. Afterward, 0.1ml of suspension was taken from the beaker, spread on Potato Dextrose Agar (PDA) plates and incubated at ambient temperature (28±2^oC) for 24 hours for colonies to develop. Sub-cultures were made to obtain pure isolates. The isolates were identified to species level by molecular characterization, physiological and morphological standard methods.

In vitro Inhibition of Fungal Isolates by Biofungicides

A modified dual culture technique after Ferreira et al.²⁷; Gwa and Abdulkadir²²; Gwa and Ekefan²⁸ was adopted. Determination of the zone of inhibition was done using the dual culture technique. A 2-day old Biofungicide isolate was streaked on one side of PDA or NA in a 9cm Petri dish and each pathogen was inoculated at 5cm away at the opposite side and incubated at 28±2^oC. Three replicates of each were made. Control was prepared by inoculating only the pathogen on PDA or NA without being challenged. Percentage inhibition of fungal growth was calculated.  The zones of inhibition were measured using a well-calibrated transparent meter rule and recorded.

In Vivo Effects of Biofungicides on Rot Pathogens of Yam Tubers

As part of the precautionary measures to reduce contamination, the healthy yam tubers were washed to remove soil particle and debris. The tuber was rinsed with 10% concentration of sodium hypochlorite for 5 minutes. This was further cleaned with sterile distilled water and sterilized with 70% ethanol. The healthy yam tubers were carefully placed on sterile paper towels in a Laminar Air Flow Cabinet for ease of drying. The methods of Gwa and Ekefan28 were adopted. Treatments comprising the pathogenic fungal isolates, each paired with four biological antagonists (Biofungicides) were set up to determine the effect on rot in yam tubers. Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, and Pseudomonas syringae served as the antagonist while the fungal isolates served as the control. Each of the Biofungicides was matched with the pathogenic fungal isolate and the yam tubers were inoculated separately.For each treatment, one litre of the pathogenic fungi in liquid medium was mixed with one litre of the biological antagonist in a liquid medium and stirred for 5 minutes. A pin bar (sterile) was used to scratch each yam tuber, starting from the proximal end to the distal end. The injured yam tuber was completely submereged in the dispersed medium for 10 minutes. The yam tubers were then removed and transferred into a moist white nylon bag. To create a conducive environment for the test fungi/pathogen,  the nylon bags were tied with a rubber band and punctured. The nylon bags containing the inoculated yam tubers were placed on benches at room temperature for 42 days. The control experiment was set up with yam tubers dipped in pathogenic fungi in a liquid medium.The treated tubers were checked for rot by cutting it at the point where it was injured. To ascertain the degree or rot/damage, comparison was made with the control. The percentage inhibition was calculated according to the method Whipps²⁹

Percentage inhibition = R₁ – R₂   x 100%

                                          R₁

Where; R₁ is the furthest radial distance of pathogen in control tubers

             R₂ is the furthest radial distance of pathogen in antagonist-incorporated tubers.

Statistical Analysis of Data

The statistical analyses used in this research work were based mostly on methods in the standard textbook, Wahua³⁰, and a computer package, Statistical Product and Service Solutions (SPSS). For each treatment, each outcome was replicated thrice using triplicate analysis. Data obtained in this study was presented as mean±standard deviation on the tables. Data were also subjected to Analysis of Variance (ANOVA). Statistical comparisons were done by using one-way ANOVA followed by Ducan’s Multiple Range Test (DMRT) using the IBM (SPSS software, version 22). The level of significance was set at P<0.05 (probability level).

RESULTS

A total of five fungi namely- Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, Aspergillus flavus and Penicillium sp were repeatedly isolated from the rot-infested tissues of the yam tuber samples. A. niger had the highest frequency of occurrence of 76.00%. This was followed by F. oxysporum with 60.00% occurrence, while A. flavus had the least frequency of occurrence of 36.00% (Table 1). Pathogenicity test showed that all the five fungi identified: A. niger, F. oxysporum, R. stolonifer, Penicillium spp, and A. flavus were all pathogenic. The most virulent amongst the fungi was A. niger with 100.00% rot followed by A. flavus with 88.00% rot while the least was Penicillium sp with 50.00% rot (Table 2).

Comparing the radial growth of pathogenic fungi; A. niger, F. oxysporum, R. stolonifer, Penicillum sp, and A. flavus was higher in the control than when paired with the Biofungicides. Results show that Bacillus subtilis inhibited the growth of A. flavus with a mean zone of inhibition of 41.33mm followed by F. oxysporum with a mean zone of inhibition of 38.70mm and the least was Penicillium sp with 17.80mm mean zone of inhibition. This showed that Bacillus subtilis was effective on the inhibition of the growth of all five pathogenic test fungi and its inhibitory effect was significantly (P˂0.05) different from the effects of the other Biofungicides. The inhibitory effects of Trichoderma viride showed that it reduced the growth of R. stolonifer with a mean zone of inhibition of 34.70mm, followed by A. flavus with 28.40mm mean zone of inhibition, while the least was Penicillium sp with 15.70mm mean zone of inhibition (Table 3). Trichoderma harzianum showed a reduction in the growth of A. flavus with 25.66mm mean zone of inhibition, followed by its inhibition of F. oxysporum with a mean zone of inhibition of 18.74mm, while the least inhibited pathogen was A. niger with 9.82mm mean zone of inhibition. The least effective of all four Biofungicides was Pseudomonas syringae which inhibited the growth of A. flavus with a mean zone of inhibition of 27.10mm, followed by A. niger having 13.50mm mean zone of inhibition, while the least was R. stolonifer with 4.50mm mean zone of inhibition (Table 3).

The in vitro results of the dual culture of the Biofungicides and the test pathogenic fungi showed that when the mycelium of both cultures came in contact with each other, the hyphal growth of the test pathogenic fungi was found to be inhibited by the hyphae of the four Biofungicides. The result of the dual culture also shows that the Biofungicides grew much faster than the pathogen, parasitized on the test pathogen and deprived it of absorbing the nutrients from the substrates. The test pathogenic fungi eventually died. The results of the dual culture indicated that the Biofungicides inhibited the growth of the pathogenic test fungi at varying degrees during the incubation period. B. subtilis inhibited the growth of A. flavus significantly (P˂0.05) with 48.67% mean percentage growth inhibition and the least inhibited was Penillium sp at 13.16% (Table 4). T. viride inhibited the growth of R. stolonifer significantly (P˂0.05) when paired together with 40.16% mean percentage growth inhibition, followed by A. flavus at 30.64% while the least growth inhibition was recorded by Penicillium sp at 12.95% when paired with T. Viride. Pseudomonas syringae inhibited the growth of A. flavus significantly (P˂0.05) with 31.10% mean percentage growth inhibition when paired together, followed by A. niger at 12.36% and the least inhibited was R. stolonifer at 2.52% mean percentage growth inhibition (Table 4).

This study showed that all four Biofungicides were significantly (P˂0.05) effective in reducing rot in the yam tubers treated in vivo. Reduction in rot ranged from 52.55-83.40mm in A. niger when paired with the four Biofungicides. In R. stolonifer, the rot reduction ranged from 61.02-92.60mm. In F. oxysporum, the rot reduction ranged from 51.60-78.59mm. In A. flavus, rot reduction ranged from 44.65-63.40mm. Reduction in rot ranged from 77.80-81.27mm in Penicillum sp when paired with the Biofungicides (Table 5). Reduction of rot indicates a decrease in the radial growth of the pathogenic test fungi. Bacillus subtilis reduced the rot of treated yam tubers significantly (P˂0.05) more than the other Biofungicides.

DISCUSSION

The fungi consistently isolated from the rot-infested tissues of the susceptible variety of white yam tuber samples agrees with the report of Anuagasi et al.²⁴ who isolated similar microorganisms in association with forestry nursery seedlings. These microorganisms have been associated with post-harvest rot in yam according to reports by Amusa et al.¹¹; Okigbo and Odurukwe²⁶; Gwa et al.¹⁶; Gwa and Ekefan²⁸. These organisms infected the yam tubers during the pre-harvest stage while in the soil and also through natural openings and physical damages incurred by the tuber during post harvest operations and then manifests fully during storage^{7, 11}. This is similar to the observations made in this study. Pathogenicity test revealed that fungi isolated in this study were pathogenic. This can be attributed to the potential of the pathogen to utilize the nutrients in the tubers for growth, development and other metabolic avctivities.

Various methods of control of post harvest rot of yam tubers such as the use of plant extracts have been reported by various workers such as Amusa et al.¹¹; Okigbo and Odurukwe²⁶; Anukwuorji et al.¹². However, the use of plant extracts has its short comings and is limited in its function to inhibit the growth of pathogenic microorganisms. The use of plant extracts is cumbersome to handle and the extraction medium usually interferes with its inhibitory potentials. Hence, the use of Biofungicides to control yam rots in this study because of their antagonistic properties and proven potentials to inhibit the growth of microorganism using antagonism. In this present study, Biofungicides of Trichoderma harzianum, Trichoderma viride, Bacillus subtilis and Pseudomonas syringae were used to control the pathogens inducing rot in white yam and these produced a significant inhibition on the growth of post harvest yam rot fungi. This observation is in tandem with the works of Okigbo and Ikediugwu¹⁹; Okigbo and Emeka¹³; Gwa and Ekefan²⁸.

It was observed that the faster rate of development of rot amongst tubers was probably related to the elevated frequency of occurrence of the pathogens. This observation is in sync with reports of Okigbo and Ikediugwu¹⁹ who reported the prevalence of A. niger which maintained a higher frequency of occurrence over a more prolonged duration. The in vitro results of the dual culture of the Biofungicides and the test pathogenic fungi showed that when the mycelium of both cultures came in contact with each other, the hyphal growth of the test pathogenic fungi was found to be inhibited by the hyphae of the Biofungicides. The results of the dual culture indicated that the Biofungicides grew faster than the pathogens, parasitized on the test pathogen, and deprived it of absorbing the nutrients from the substrates. This observation is in tandem with Okigbo and Emeka¹³; Gwa et al.¹⁶; Gwa and Ekefan²⁸ who observed the same in their studies.

T. harzianum and T. viride inhibited the growth of all pathogenic test fungi through their ability to grow much faster than the pathogenic test fungi thus, competing efficiently for space and nutrients. This has been reported by Barbosa et al.³¹; Siameto et al.³²; Mokhtar and Aid³³ on the production of both non-volatile and volatile antibiotics by species of Trichoderma. These substances produced by Trichoderma sp served in the biological control of storage rot of yam tubers. Bacillus subtilis inhibited the growth of all test pathogenic fungi significantly when paired together. This observation is in tandem with Okigbo¹⁷; Okigbo³⁴ in which B. subtilis displaced naturally occurring mycoflora on the surface of yam tubers or caused a reduction percentage of rot in treated tubers. Pseudomonas syringae slightly inhibited the growth of the test pathogenic fungi when paired together. This observation is in sync with the report of Okigbo and Emeka¹³. In other studies, the saprophytic strain of the bacterium, Pseudomonas syringae (L-59-66) also satisfactorily controlled the difficult grape rots (Botrytis cinerea) and blue mold of citrus (Penicillium citrinum)³⁵. Arya³⁵ reported that this saprophyte has been developed into a commercial brand (Ecosuinex).

CONCLUSION

The results of this study showed that these Biofungicides; Trichoderma harzianum, Trichoderma viride, Bacillus subtilis and Pseudomonas syringae have potentials to control rot in post harvest white yam tubers. Overall, Bacillus subtilis was recorded as the most effective in controlling the pathogenic test fungi. Thus, the use of Biofungicides as biocontrol agents is an economically viable way of suppressing post harvest rot of white yam. This can provide alternative ways in reducing rot in yams than the use of chemical fungicides.

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