The experiment was conducted in the Biological Science Laboratory, Federal University, Dutsin-Ma, Local Government Area (LGA) in Katsina State, Nigeria (latitude 12° 27’ 18’’ N and longitude 07° 29’ 29’’ E) in 2016.
Diseased samples of tomato fruits showing various rot symptoms were collected from three locations of Dutsin-ma Local Governments Area in Katsina State namely: Darawa, Dutsin-Ma and Makera at forth nightly interval and the samples were packaged in sterile polythene to prevent to prevent further attack by insects and pathogens.
Diseased samples were washed under running tap water after which the samples were chopped into small pieces of about 2-3 mm in diameter and kept in a sterile Petri dish. The pieces were dipped into 5% hypochlorite solution for about 20 seconds. The pieces of the tomatoes were transferred into Petri dishes containing sterile distilled water and were washed thoroughly in three successive changes of sterile distilled water. About 15ml of the molten PDA was poured in Petri dishes of 9 cm in diameter and allowed to solidify. After solidification, four pieces of the diseased tomato tissues were aseptically placed at different distance in the Petri dishes. The Petri dishes were tightly covered with a masking tape to prevent contamination by air-borne pathogens. The dishes were incubated at ambient room temperature (33°C-37°C) for 7 days to allow for the growth of fungi.
The pathogens that grew on the Petri dishes were sub-cultured after incubation period of 7 days to have a pure culture of the isolates. This was done by transferring the fungi mycelial on agar plates containing the medium by using an inoculation needle to place the mycelial at the centre of the Petri dishes. The Petri dishes were tightly sealed with a masking tape and thereafter incubated for 7 days. When growth was established, growth patterns were determined based on microscopic and morphological characteristics and were compared with existing authorities (Ahmed and Ravinder, 1993; Burgess., et al. 2008). Test fungi in this study were Rhizoctonia solani and Fusarium oxysporum which were mostly isolated in the locations.
Records of the organisms isolated were kept on periodic basis to determine the frequency of occurrence of the isolates. Since isolation and characterization were carried out at fortnightly interval, the number of times each fungal pathogen was isolated at fortnightly interval was expressed as a percentage of the total of all the different fungal organisms isolated over the period (Okigbo and Ikediugwu, 2000), which was calculated as follows:
x = number of times an individual isolate has occurred over the period
n = total number of fungal organisms isolated in the study area over the period
Fresh and healthy-looking tomato fruits were collected from the markets and were surface sterilized by dipping them in 5% sodium hypochlorite for 20 seconds and then rinsed with four successive changes of sterile distilled water. The healthy-looking fruits were then wounded with a sterile needle. Mycelial disc from R. solani and F. oxysporum were carefully lifted from the pure culture of the respective plates and introduced directly into the wounded tissues of healthy tomato fruits. In another experiment, three tomato fruits were each surface sterilized and wounded with a sterile needle and inoculated separately with sterile distilled water instead of the mycelial of R. solani and F. oxysporum. This served as the control experiment for both R. solani and F. oxysporum. The inoculated fruits and the control were placed separately lined with a moist filter paper and cover with aluminium foil and incubated at ambient room temperature (33°C-37°C). The fruits were observed for symptoms of rot development after five days of incubation.
The methods of Gwa and Akombo, (2016) and Gwa and Nwankiti, (2017) were adopted for this experiment. Accurately, 40g, 80g and 120 g of powdered leaves of neem (A. indica) and rhizomes of ginger (Z. officinale)were measured respectively using an electric weighing machine. Sterile water was heated using hot plate to a temperature of 100oC. 1-litre of the sterile distilled water was each measured using a measuring cylinder and was poured into conical flasks containing the neem leaves powder and ginger rhizome powder at different level of concentrations respectively. The mixtures were vigorously stirred and left to settle for 24 hours, after which they were filtered through three layers of muslin cloth. Concentrations of 40 g/L, 80 g/L and 120 g/L of the neem leaves and ginger rhizomes were prepared accordingly. 5 ml each of the prepared plant extracts at the different level of concentrations were used to amend in 15 ml of potato dextrose agar.
The method of Amadioha and Obi (1999) was used to measure the fungitoxicity of the extracts. This method involved direct treatment of potato dextrose agar (PDA) medium with plant extracts before inoculation of fungus. This involved creating four equal sections on each plate by 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 at different levels of concentration were poured into Petri dishes containing the media separately, 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) which was obtained from one-week old cultures of R. solani and F. oxysporum. Three dishes were plated with extract of each plant at different concentrations. The control experiment had 5 ml of sterile distilled water added to the PDA plates in place of plant extracts respectively, the treatment and control plates were replicated three times and were incubated for 4 days at ambient room temperature (33°C-37°C) and measurement of mycelial radial growth as radius of growing fungal colony were undertaken at intervals of 1 day for 4 days using a transparent ruler. The absence of growth in any of the plates was indication of the potency of the extract against the test fungal. Fungitoxicity was determined as percentage growth inhibition (PGI) according to the method described by Gwa and Akombo, (2016).
R = Distance of fungal growth from the point of inoculation to the colony margin in the control plate
R1 = Distance of fungal growth from the point of inoculation to the colony margin in treated plate.
Data collected were subjected to Analysis of variance (ANOVA) using GenStat Discovery Edition 12 for ANOVA and means separation, Minitab Release 17 for descriptive statistics and Graph Pad Prism 6 for trend graphs. Statistical F-tests were evaluated at P ≤ 0.05. Differences among treatment means for each measured parameter were separated using Fisher’s least significant difference (FLSD) (Cochran and Cox, 1992).
Results presented in Plates I shows some cultures and photomicrographs of the fungi pathogens isolated from tomato fruits. Results presented in Figure 1 show the frequency of the fungi organisms that were isolated and identified in the different locations. F. oxysporium, F. monilliforme, A. niger, A. flavus and R. solaniwere identified. Among the pathogenic fungi, F. oxysporum was the most frequently occurring fungus constituting 67% in Darawa, 51.36% in Dutsin-ma and 33.30% in Makera. A. niger was the second most encountered pathogen constituting 4.17% in Darawa, 21.01% in Dutsin-ma, and 31.85% in Makera. The percentage frequency of occurrence of A. flavus was 13.69%, 4.17%, and 28.10% in Darawa, Dutsin-ma and Makera regions, respectively. R. solani recorded percentage frequency of 9.52% in Darawa, 12.94% in Dutsin-ma, and 2.22% in Makera. The least occurred isolate was F. monilliforme with percentage frequency of occurrence of 4.17% in Darawa, 10.50% in Dutsin-ma and 4.44 % in Makera. Fungi pathogenic organisms were identified based on their morphological growth patterns as well as microscopic characteristics and were compared with existing authorities
|A.flavus||1.00 ± 0.57||0.33 ± 0.03||2.00 ± 0.57||0.14ns|
|A.niger||0.33 ± 0.03||2.33 ± 0.88||3.00 ± 1.00||0.12ns|
|F.monilliformes||0.33 ± 0.03||2.33 ± 0.20||0.66 ± 0.06||0.62ns|
|F.oxysporum||3.33 ± 1.33||5.66 ± 0.33||3.67 ± 1.86||0.45ns|
|R.solani||0.66 ± 0.06||2.00 ± 1.53||0.33 ± 0.03||0.48ns|
|Total||5.67 ± 1.86a||12.33 ± 3.38b||9.67 ± 2.91ab||0.04|
Table 1: Number of fungi pathogens isolated from different locations.
The study revealed that the fungi organisms isolated from the infected tomato fruits were pathogenic on the healthy tomato fruits. A. niger was more virulent where the inoculated fruits were completely rotten at the end of the fifth day of incubation. The fruits were completely disintegrated with extensive mycelial growth forming a dark colour covering the fruit skin. Fruits inoculated with F. oxysporum had water-soaked lesions with some white to pink mycelial while fruits inoculated with A. flavus had whitish cheesy like lesions. Samples inoculated with F. monilliforme had small water soaked lesion with slightly brownish appearance on the inoculated areas while tomato fruits inoculated with R. solani had small hard dark lesion around the inoculated area. The fruits that were not inoculated with any fungi pathogen however, showed no signs and symptoms of rot.
The result presented in Table 2 revealed that the plant extracts inhibited the growth of test fungi, although the rate of inhibition varied with different extracts and concentrations used. However, growth inhibition in all the test pathogens took a similar trend in all extracts as the concentration of the tested plant extracts were found to increase with increase in the concentration as incubation period increased. The highest concentration of 120 g/L was the most potent and had the highest inhibition on the pathogens. At concentration of 120 g/L, Z. officinale produced lower effect on R. solani compared with A. indica. The effectiveness of the extracts differed significantly (P ≤ 0.05) comparing them at 120g/L on mycelial growth inhibition of R. solani. The effectiveness of neem and ginger extracts did not differ significantly (P ≤ 0.05) at 40g/L and 80g/L. A. indica inhibited the mycelial growth of R. solani recording percentage growth inhibition of 44.90% compared with Z. officinale which inhibited the growth of R. solani by 43.38%. Neem crude plant extract was the most effective on R. solan but less effective on F. oxysporum. There was however, no significant difference (P < 0.05) between the extracts at each level of comparison when they were tested on F. oxysporum (Table 3).
|Concentration (g/L)||Plant Extract||df||T-Value||P-Value|
|A. indica||Z. officianale|
|40||44.90 ± 11.80||43.38 ± 6.88||17||0.11||0.91|
|80||41.07 ± 9.27||61.84 ± 6.49||19||1.84||0.08|
|120||97.22 ± 2.78||63.31 ± 6.79||14||4.62||< 0.01*|
Table 2: Percentage Growth Inhibition of R. solani at different levels of Concentrations of A. indica and Z. officinale.
|Concentration (g/L)||Plant Extracts and Growth Inhibition (%)||df||T-Value||P-Value|
|A. indica||Z. officianale|
|40||37.51 ± 6.97||22.91 ± 4.92||19||1.71||0.10|
|80||40.56 ± 6.30||35.00 ± 7.24||21||0.58||0.56|
|120||45.48 ± 6.79||42.73 ± 6.67||21||0.29||0.77|
Growth of F. oxysporum on potato dextrose agar amended with Z. officinaleis presented in figure 2. The results indicated that mycelial extension was higher in the control (0 g/L of Z. officinale). This study reveals that the radial growth decreases with increase in the concentration of ginger. At 120 g/L the radial mycelial growth was lowest compared to 80 g/L and 40 g/L respectively. Results presented in figure 3 revealed the growth inhibition of F. oxysporumthroughout the period of incubation. It showed that radial mycelial growth increased with increase in incubation period but decreased with increase in concentration. At 120 g/L the radial mycelial growth was lowest compared to 80 g/L and 40 g/L.
The authors declare that there is no conflict of interest regarding the publication of this paper.
This research received no specific grant from any funding agency in the public, commercial or not-for- profit sectors
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