Abstract: Background and Objective: Sesame (Sesamum indicum L.) is one of the most important oil crops in Ethiopia next to coffee arabica. The present study was aimed to evaluate and characterize the potential biocontrol agents of Trichoderma species isolates against 2 pathogenic fungi viz., Alternaria isolate (AUA1) and Fusarium isolate (AUF5) under in vitro condition. Materials and Methods: Infected leaves, seeds and soil samples of sesame were collected from different sesame farms of Wolkait district, Northern Tigray, for the isolation of fungal pathogens. The pathogenicity test of these isolates was confirmed on detached leaves of sesame plants resulting in typical symptoms of leaf blight and wilt disease. The in vitro potential of 7 Trichoderma species isolates were evaluated against two isolates of phytopathogenic fungi in dual culture techniques through production of volatile and non-volatile inhibitors. Results: Among the Trichoderma species isolates, T-12, T-158 and T-97, exhibited a significant (p<0.05) enhancement of germination percentage in sesame seeds compared to control (55%). In vitro confrontation analysis revealed that the highest mean inhibitory effect on the growth of the test pathogens were achieved by T. asperellum T-131 (79.49%) against AUF5 and T. asperelloides T-97 (76.42%) against AUA1 while, T. viride T-33 isolate showed the lowest mean inhibition compared to control consisting of any of the two test pathogens growing alone. The highest non-volatile inhibition effect was exhibited by T. asperellum isolates (T-11 and T-12) against AUF5 isolate. Conclusion: Among Trichoderma species, excellent results were observed for T. asperellum and T. asperelloides indicating better plant growth promoting potential and thus, exhibiting perspective for their commercial exploitation.
INTRODUCTION
Sesame (Sesamum indicum L.) belongs to family Pedaliaceae is considered as one of the most ancient oil seed crops known to mankind, it is also known as Benne seed in Africa and sim-sim in East Africa1,2. The genus has many species and most of them are wild. Most wild species of the genus Sesamum are native to sub-saharan Africa3. It is extensively cultivated in the tropics and the temperate zone of the world4,5. India, China, Burma, Sudan and Mexico are the largest producers of this oil seed6. According to Raney et al.7, the global cultivated area of sesame was about 7.4 million ha producing about 3.4 million Mt which make it the 5th most important oil seed crop on an area basis worldwide. Raney et al.7 also reported that, Sudan grows about 20.12% (1.48 million ha) of the world cultivated area and contributes about 9.24% (0.32 million Mt) of the total production. Sesame is the second largest source of foreign exchange earner after coffee in Ethiopia8 as it is the major oil seed in terms of exports in Ethiopia, accounting for over 90% of the values of oil seed exports.
Pathogenic fungi are the most common and economically important foliar and soil borne diseases of sesame9. Availability of healthy seed that have tolerable levels of A. sesami10 and exclusion of the disease from non-infested sesame growing areas are some of the practical management options for controlling Alternaria Leaf Spot (ALS). However, the management options are not economical for resource-limited small-scale farmers who are the major producers of sesame in Ethiopia. In addition, Fusarium wilt of sesame caused by Fusarium oxysporum remains a challenging task in terms of management in Ethiopia11. Application of fungicides, crop rotation with non-hosts of the fungus and disinfestation of the soil to control these diseases is not economical and it is labour intensive. Besides, chemicals pose serious health hazards to an applicator as well as to a consumer of the treated material. Prospects of biological control of soil-borne plant pathogens using most promising bio control agent, the genus Trichoderma has been taken and its potential was evaluated to control fungal diseases of sesame crop under in vitro condition.
Sesame production is affected by many biotic and abiotic stresses. Among the biotic agents, fungi cause major diseases followed by bacteria, viruses and nematodes. Major sesame diseases caused by fungi are: Leaf spot/blight (Alternaria sesami)10, Fusarium wilt of sesame (Fusarium oxysporum f. spp. Sesami), Cercospora leaf spot (Cercospora sesami)12, Powdery mildew (Sphaerotheca fuliginia), stem blight (Phytophthora parasitica) and bacterial leaf blight/spot (Pseudomonas sesami)13, Mycoplasmal such as; phyllody (Mycoplasma)14, viral diseases such as; Leaf curl (Nicotina virus)15, Mosaic (Cucumber mosaic virus)16, Necrosis (Tobacco streak virus)17 and root knot nematode (Meloidogyne hapla)18.
Among these diseases, at present leaf spot/blight caused by Alternaria sesami and Fusarium wilt caused by Fusarium oxysporum are widespread and have continued to be the major constraints in the production and productivity of sesame in Ethiopia. Alternaria Leaf Spot (ALS) of sesame caused by the seed-borne fungus Alternaria sesami. Typical symptoms on infected plants include lesions with concentric rings on leaves that often coalesce to form large blighted leaf areas19. Species of Alternaria are spread from field to field and from one geographical area to another by several means, including wind-borne spores and adherence of soil to seedlings, farm equipment or animals20. In some pathosystem, viable spores pass through the digestive tract of ruminants fed on diseased plant tissue. This may be an important means of spread of the pathogen from infested to non-infested areas within a field or from infested fields to non-infested fields21. On the other hand, Fusarium wilts caused by Fusarium oxysporum f. spp. Sesami (FOS) is a devastating disease infecting sesame crop right from seedling to maturity resulting in crop losses in varying degrees depending on the severity of infection. It has been reported as a most important soil borne fungal disease in which the water-conducting (xylem) vessels become blocked, so that the plant wilts and often dies22.
The use of chemicals to control plant disease is an effective and successful approach. However, the large-scale use of chemical pesticides has resulted in problems including safety risks, environment pollution and biodiversity decrease and pathogen resistance. Hence, it is quite prudent to develop a more effective and eco-friendly strategy instead of chemical pesticides for controlling plant diseases. Biological control is supposed to be a crucial strategy for reducing disease incidence and severity by direct or indirect manipulation of antagonistic micro-organisms to protect crops from diseases without the adverse effects of chemicals23. In the literature, the use of Biological Control Agents (BCAs) has been documented as a potential alternative to control Fusarium species24 and Alternaria species25,26. In recent years Bacillus, Pseudomonas species, Trichoderma and Cryptococcus species have been the most commonly investigated micro-organisms for the control of Fusarium species regarding tests conducted in vitro or on different plants, in controlled conditions or even under field conditions. In this direction, a number of commercial products have been registered at international levels27, but there is no available bio formulated BCAs for the control of sesame diseases in Ethiopia.
The fungal genus Trichoderma contains species well documented as biocontrol agents of many crop pathogens, including Fusarium and Alternaria species28,29. Depending on species (Trichoderma species, pathogen and host plant), members of the genus Trichoderma may employ one or more modes of antagonism such as; the production of toxins (e.g., antifungals and chitinases), mycoparasitism (physical disruption of pathogen hyphal growth: coiling, penetration, dissolution of cytoplasm), induction of a host defense response or success in monopolizing rhizosphere nutrients and space30-32. In recent years, the filamentous fungi Trichoderma species have attracted much research attention because they are effective biocontrol agents against a wide range of plant pathogens.
Considering the use of Trichoderma species as control agents of Fusarium and Alternaria species, there is strong support that one or more Trichoderma species would be effective antagonists of Fusarium wilt and leaf blight of sesame33. Biocontrol potential of seven Trichoderma species isolated from different local agricultural soils against the causative agent of Fusarium wilt and leaf blight of sesame isolated from a diseased sesame farms from welkait district, Western Tigray, Ethiopia, were evaluated. Found Trichoderma species were then tested for antagonistic activity toward both fungal pathogens of sesame under in vitro conditions.
Therefore, the objective of this study was to evaluate and determine the potentiality of Trichoderma species as biocontrol agents against sesame fungal pathogens (AUA1 and AUF5 isolates) in dual culture techniques and through production of volatile and non-volatile inhibitors.
MATERIALS AND METHODS
Description of study areas: The study was conducted in Welkait district, Western Tigray, Ethiopia, from October, 2018 to September, 2019. Welkait is located 437 km west and 1220 km northwest far from the capital city of Tigray region (Mekelle) and country capital city (Addis Ababa), respectively at 13°30 00" and 14°07 00" North latitude and 36°40 15" and 37°48 00" East longitude with an altitude ranges from 677-2755 m above sea level. The district has 28 sub-districts of which 14 are with lowland agro-ecology as shown in Fig. 1 adapted from https://www.researchgate.net/figure/Map-of-the-study-area-Welkait-District-Western-Tigray-Ethiopia_fig1_ 320863247. The annual temperature and unimodal rainfall of the district are 17.5-25°C and 700-1800 mm, respectively. Welkait district is known for its fertile alluvial soil, which grows cash crops such as; sesame, cotton and sorghum.
Sample collection: Sesame leaves and seed showing visible symptoms of fungal pathogens and soil of each sesame sample were collected from three different Kebeles of Welkait woreda namely; Bet-Mulu, Kalema and Lalay Mayhumer.
| Fig. 1: | Map of the study area, Welkait district, Western Tigray, Ethiopia |
| Table 1: Trichoderma isolate identification and characteristics used in this study | |||
| Isolates | Species | Substratum | Geographic location |
| T-11 | T. asperellum | Faba bean soil | Sheno, North Shoa |
| T-12 | T. asperrelum | Faba bean soil | Fiche, North Shewa |
| T-32 | T. gamsii | Coffee rhizosphere | Gera, South west |
| T-33 | T. viride | Faba bean soil | Ambo, West Shewa |
| T-97 | T. asperelloides | Coffee rhizosphere | Jimma, South west |
| T-131 | T. asperelloides | Cotton soil | Wolkait, North Tigray |
| T-158 | T. viride | Coffee rhizosphere | Teppi, South west |
For each sampling site, sample collections were made from three fields located within 15-20 km distance19. The collection sites represented different geographic locations at varying elevations and with different climatic conditions. Visual observations were made for the manifestation of the typical symptoms on field growing sesame plants and the leaf samples were in kaki paper bags, endorsed with relevant information and brought to the laboratory for further studies. The top layer of a soil litter and the upper soil horizon (4-6 cm) was discarded and 100 g of soil from approximately 10-15 cm depth was collected, placed in polyethylene bags and labeled. Moreover, defect sesame seeds were also collected in kaki paper from each study site. The isolation and identification of fungal pathogens were conducted at the Department of Microbial, Cellular and Molecular Biology, College of Natural and Computational Sciences, Addis Ababa University (AAU), Addis Ababa, Ethiopia.
Isolation of sesame fungal pathogens: Ten grams of soil samples (if necessary pulverized by means of a mortar and pestle and passed through a 0.5 mm soil screen mesh to remove large debris and root fragments) were suspended in 90 mL sterile distilled water and thoroughly mixed. A 10 mL aliquot was then used to prepare a series of dilutions in the range of 10–1-10–3 and inoculated onto Czapek Dox Agar (CDA) and supplemented with streptomycin (250 mg L–1) to prevent bacterial growth. Three replicates were done for each medium and soil sample. Plates were incubated at 25°C for a period of 3-5 days and examined daily for colony development. Sesame pathogens were subsequently grown on PDA medium and sub-cultured to obtain pure culture5.
Samples of infected sesame leaf specimens showing typical symptoms of fungal pathogen and seeds were washed thoroughly with distilled water and blot dried in the wood cabinet. The leaves were cut with a sharp sterilized blade into small bits (1 cm2), keeping half healthy and half diseased portion intact. Then, leaves and seed were surface sterilized with 2% of sodium hypochlorite (NaClO) solution for 30 sec, then rinsed in three sequential sterile distilled water in Petri plates to remove traces of NaClO and again blot dried in the wood cabinet5,31. Later, leaf bits and seeds were inoculated aseptically on autoclaved and cooled Potato Dextrose Agar (PDA) medium in sterilized Petri plates under Laminar-air-flow cabinet and incubated at 25°C. Through frequent sub-culturing, the test pathogens were purified and the pure cultures were maintained on a PDA slant in glass test tubes at 4°C for further study.
Identification of sesame fungal pathogens: Sesame fungal pathogens were identified according to their growth and cultural characteristics, morphological and microscopical features. The sesame fungal pathogens were characterized based on the following parameters such as; pigment production, colony color, spore or conidia producing structures and spore shapes. Spore and mycelium characteristics were studied under compound microscope of 400x magnification (Olympus Microscope, Germany). These characteristics were used in identifying the fungal isolates to the genus level following standards described by Mathur and Kongsdal34.
Slide culture technique: A clean slide was placed in 9 cm diameter plates and then a small amount of autoclaved melted PDA medium was spread over the slide to make a thin PDA film. Five millimeter disc of the isolated pathogens were placed on slide. Distilled water was poured in Petri plates to avoid drying (incubated at 28+2°C for 3-5 days). The area was observed microscopically (Olympus Microscope, Germany) by staining with lactophenol cotton blue35.
Sources of Trichoderma species: Trichoderma species were obtained from Mycology Laboratory, Department of Microbial, Cellular and Molecular Biology, College of Natural and Computational Sciences, Addis Ababa University (AAU). Seven Trichoderma species isolates namely two Trichoderma asperellum isolates (T-11 and T-12), two Trichoderma viride isolates (T-33 and T-158), two Trichoderma asperelloides isolates (T-97 and T-131) and Trichoderma gamsii (T-32) were used in this study. The identifications and details are listed in Table 1. These isolates were chosen for this study because several reports demonstrated their high antagonistic in different pathosystems36,37. The isolates were maintained on Potato Dextrose Agar (PDA).
Pathogenicity test: To determine pathogenicity, two isolates of the species and 2-month-old sesame plants (cultivar) were grown in a greenhouse at 25±2°C. Inoculation was performed by spraying conidial suspensions of 1×105 spores/mL on plants, without wounding. The conidia were scraped from the 10-day cultures on V8 agar plates grown at 25°C with a 12 h photoperiod of UV light38. Control plants were sprayed with sterile water. The inoculated plants were first kept in humid plastic bags for 2 days at 25°C, then the cover was removed and the plants were transferred to a glasshouse at 25 ±2°C.
Effect of Trichoderma metabolites on sesame seed germination: The effect of the culture filtrate of each Trichoderma isolates on the sesame seed germination was investigated. The inoculum preparation was carried out on potato dextrose broth medium. For inoculation with Trichoderma isolate, 10% of spore suspension at a concentration of 105 spore/mL was used. After incubation under shaking at 150 rpm at 25°C for 6 days, the culture broth was filtered using a filter paper. The filtrate was centrifuged at 5000 rpm for 15 min, the supernatant was collected and the pellets were discarded. In this respect, sesame seeds were soaked for 24 h in each of the prepared Trichoderma filtrates. Untreated seeds served as a control. The seeds were dried for 1 h in a laminar flow cabinet. For each treatment, 960 seeds were plated (40 seed/plate) on wet blotters and then incubated at 25°C for 1 week. Three replicates were used for each treatment. After which, the germination (%) was calculated39:
Characterization of different bio-control mechanisms of Trichoderma isolates
Antagonistic assessment of Trichoderma species: The invasive growth of Trichoderma isolates against Fusarium species and Alternaria species were evaluated using dual confrontation techniques40-43 with slight modifications. A mycelial agar plug (5 mm diameter) of each Trichoderma isolates taken from the edge of actively growing 7 days-old culture was paired against same sized mycelia disc of the test pathogens at equal distances opposite to each other in 90 mm diameter Petri plates containing 20 mL PDA. The PDA plates inoculated only with Trichoderma isolates or Fusarium species and Alternaria species served as control. There were six dual-cultures (replicates) for each Trichoderma-Fusarium species and Trichoderma-Alternaria species combination. The Trichoderma species were grown on PDA in 90 mm diameter Petri plates until they completely colonized the plate. The growth of the pathogen in both test and control experiments were recorded according to the method by Dennis and Webster44. The Percentage Inhibition of Radial Growth (PIRG) was computed as follows:
Where:
| R1 | = | Radial growth of pathogen in control |
| R2 | = | Radial growth of pathogen in dual confrontation experiments with antagonists |
The antagonistic effect of Trichoderma isolates was assessed in semi-quantitative means; >75 PIRG indicating very high antagonistic activity, 61-75 PIRG indicating high antagonistic activity, 51-61 PIRG, indicating moderate antagonistic activity, <50 PIRG indicating low antagonistic activity and 0 indicating no activity. A Clear Zone of Inhibition (CZI) was also determined by measuring the clearance between the colony margins of the Fusarium and Alternaria species and Trichoderma isolate.
Effect of volatile metabolites: Selected Trichoderma isolates based on the mycelium inhibition assay against sesame fungal pathogens were evaluated for the production of volatile inhibitory substances under in vitro conditions following the modified methods of Dennis and Webster44. Five millimeter disc of Trichoderma colony was inoculated centrally in Petri plates containing PDA medium in triplicates. The Petri plates were sealed at the edges and incubated at 25°C. After 24 h, the test pathogen was inoculated on fresh PDA and the lids of the Petri plates inoculated with antagonist were replaced by the pathogen on PDA. The plates were fixed with parafilm and incubated for another 6 days, whereas, control plates were inoculated with pathogen alone45. The radius of the test pathogens will be recorded and the Percentage Inhibition of Radial Growth (PIRG) was determined after 10 days of incubation by using the same formula as described in dual culture plate testing.
Effect of non-volatile metabolites: The production of non-volatile substances by the Trichoderma isolates against the test pathogens were studied using the method described by Dennis and Webster44 and Dubey et al.45. Trichoderma isolates were inoculated in 100 mL sterilized Potato Dextrose Broth (PDB) in 250 mL conical flasks and incubated at 25°C on a rotatory shaker set at 100 rpm for 15 days. The control flasks were not inoculated with any of the culture. The liquid culture was filtered through filter paper for removing mycelia mats and then sterilized by passing through 0.45 μm pore biological membrane filter46. The filtrate was added to molten PDA medium (at 28°C) to obtain a final concentration of 10% (v/v). Heat sterilized filtrate (5 mL) was mixed with 100 mL PDA, poured in Petri plates and 5 mm diameter culture disc of test pathogen was inoculated at the center and incubated at 28°C for 10 days. There were three replicates of each treatment. The observation was taken and the mycelial growth inhibition (%) was recorded and calculated in relation to the growth of the controls.
Statistical analysis: Statistical analysis was performed using completely randomized analyses of variances (ANOVA) that was used to compare the biocontrol efficacy of Trichoderma isolates and means separated by Fisher’s protected least significant difference. The significance of effects of Trichoderma on growth characteristics was determined by the magnitude of the F-value (p<0.05).
RESULTS
Isolation and morphological identification of sesame fungal pathogens: It is clearly indicated from this study that out of 45 samples collected only 12 fungal isolates were isolated (Table 2). Among 12 fungal isolates, five isolates of were Alternaria species, six isolates were Fusarium species and Cercospora species were isolated from sesame seeds, leaves and soils collected from Wolkait district, northern Tigray. The fungal pathogen isolates were characterized depending on their morphological and microscopic characters reported in various identification manuals and categorized into three genera (Table 2). It was observed that the majority of fungal pathogens were obtained from Lalay Mayhumer followed by Bet-Mulu and Kalema (Table 2). Quantitatively two isolate groups (AUA-1 and AUF-5) were found most frequently and selected for further study.
Pathogenicity test: Symptoms first appeared about 2 days after AUT1 inoculation, lesions with chlorotic halos were developed and enlarged over time. These symptoms were similar to those observed in naturally infected plants. The same fungus was consistently isolated from diseased areas, but not from healthy controls. Thus, it concluded that the isolates were pathogenic to sesame. Inoculation experiments were conducted twice and provided similar results. Based on conidial morphology, the pathogen was identified as Alternaria sesami. Disease symptoms gradually spread from leaf margins to the mid veins. At a later stage, blight extended to the center of the leaves. The symptoms of leaf blight found on the leaves were initially round to irregular, brown colored necrotic spots with concentric zonation demarcated with brown lines inside the spots on the upper surface. In severe infection, several spots would coalesce together involving a major portion of the leaf blade and the affected leaves would dry and usually drop off.
| Table 2: List of fungal genera isolated from infected leaves, soils and seeds of sesame plants | |||
| Isolate designation | Identified genera | Sub-stratum | Location |
| AUA-1 | Alternaria species | Leaf, seed | Bet-Mulu, Korarit |
| AUA-2 | Alternaria species | Seed, soil | Lalay Mayhumer |
| AUA-3 | Alternaria species | Seed | Lalay Mayhumer |
| AUA-4 | Alternaria species | Soil | Kalema |
| AUF-5 | Alternaria species | Soil | Lalay Mayhumer, |
| AUF-6 | Fusarium species | Seed | Lalay Mayhumer |
| AUF-7 | Fusarium species | Leaf, soil | Kalema, Bet-Mulu |
| AUF-8 | Fusarium species | Seed | Bet-Mulu, Korarit |
| AUP-9 | Fusarium species | Leaf, seed | Bet-Mulu |
| AUP-10 | Fusarium species | Leaf | Kalema, Bet-Mulu |
| AUP-11 | Fusarium species | Leaf | Korarit |
| AUC-12 | Cercospora species | Leaf | Kalema, Bet-Mulu |
| Table 3: Culture and microscopic characteristics of pathogen isolates (400x) | |||||
| Morphological and cultural characteristics | |||||
| Fungal pathogens | Isolate |
Spore size (μm) |
Colony color (front) |
Conidia shape |
Mycelial shape |
| Alternaria isolate | AUA1 |
80 |
Blue black |
Oval |
Raised, no rings |
| Fusarium isolate | AUF5 |
90 |
Creamy |
Falcate |
Raised with rings |
| Fig. 2(a-d): | Colony morphology of selected sesame fungal pathogens, Fusarium isolate (AUF5), (a) Pure culture, (b) Chlamydospores (A2), Alternaria isolate (AUA1), (c) Pure culture and (d) Chlamydospores |
| Fig. 3: | Effect of Trichoderma isolates on seed germination |
| Fig. 4(a-d): | Typical dual confrontation assay that was used to quantify the interactions of 7 Trichoderma isolates with pathogen of Alternaria isolates, (a) Control plate with Alternaria isolates, (b) Reverse colony dual confrontation of Alternaria isolates (indicated by arrows) with Trichoderma, (c) Front surface colony dual confrontation of Alternaria isolates and (d) Mean relatively sensitive of Fusarium and Alternaria isolates to Trichoderma isolates |
On the other hand, the symptoms of Fusarium wilt were shown with yellow and dropped leaves, often starting on one side and stunting of the plant. Disease symptoms initiated at the bottom of the stem and progressed upwards, causing the leaves to wilt, wither and die.
Cultural and microscopic characteristics of sesame fungal pathogens: The sesame pathogens were culturally and microscopically characterized based on the size, color and shape of mycelia and conidia (Table 3 and Fig. 2).
Effect of Trichoderma metabolites on sesame seed germination: Results showed that all the Trichoderma species isolates were found effective to enhance the germination percentage compared to control (Fig. 3). However, among the seven Trichoderma species isolates, T. asperellum T-12 (93%), T. viride T-158 (91.6%) and T. asperelloides T-97 (90.8%) exhibited a significant (p<0.05) enhancement of germination (%) in sesame seeds under in vitro conditions (Fig. 3), while control treated with sterilized water significantly decreased these values (55%).
Characterization of different bio-control mechanisms of Trichoderma isolates
Antagonistic activities using a dual culture test: Using a dual confrontation assay, it was found that all the Trichoderma species isolates exhibited antifungal activity against the test pathogens of sesame crop (Table 4). By comparing and contrasting the data obtained from the dual confrontation assays, it was found that all isolates inhibited the mycelial growth of AUA1 isolate within the range of 69.25-76.42% and 66.26-79.49% against AUF5 (p<0.05). Often, Trichoderma species isolates grew within 6 days and invade both the test pathogens colony tested. The highest-level growth inhibition of AUA1 isolate was significantly restricted by T. asperelloides T-97 (76.42%), whereas, the lowest inhibition growth was recorded in T. viride T-33 (69.25%) (p<0.05). On the other hand, it was observed that the highest growth inhibition of AUF5 isolate was obtained by T. asperellum T-131 (79.49%) and the lowest inhibition was recorded in T. viride T-33 (66.26%) (Table 4 and Fig. 4).
| Table 4: In vitro evaluation of Trichoderma isolates against AUA1 and AUF5 isolates by dual confrontation culture technique, volatile and non-volatile compounds | |||||||
| Alternaria isolate (AUA1) | Fusarium isolate (AUF5) | Scale of |
|||||
| Trichoderma | Volatile |
Non-volatile |
Volatile |
Non-volatile |
antagonistic |
||
| isolates | Dual culture |
compound |
compound |
Dual culture |
compound |
compound |
activity |
| T-11 | 74.32±4.06ab |
77.90±6.1ab |
88.77±7.7a |
76.12±6.20ab |
76.20±6.7ab |
90.12±6.04a |
+ + + + |
| T-12 | 74.82±4.20a |
71.47±10.2b |
82.05±13.00 |
71.07±6.50cd |
72.80±12.6b |
90.12±6.18a |
+ + + |
| T-32 | 72.71±4.82ab |
66.50±14.65b |
16.38±17.01d |
68.78±7.73cd |
66.50±14.6b |
55.79±14.99c |
+ + |
| T-33 | 69.25±4.83b |
65.80±20.52b |
25.25±21.49d |
66.26±9.32d |
65.80±20.5b |
58.86±16.56c |
+ + |
| T-97 | 76.42±3.68a |
93.65±2.4a |
87.32±7.52a |
71.86±6.86cd |
93.65±2.4a |
87.33±7.52a |
+ + + + |
| T-131 | 75.82±4.81a |
92.16±3.8a |
71.59±11.61bc |
79.49±3.26a |
92.20±3.8a |
71.59±11.61b |
+ + + + |
| T-158 | 69.50±5.12b |
44.17±8.61c |
65.73±4.21c |
72.67±5.79bc |
44.20±8.69c |
65.74±4.21b |
+ + |
| Total mean±Sd | 73.30±5.08 |
67.70±9.5 |
59.61±32.43 |
69.04±16.91 |
69.72±24.3 |
70.85±22.88 |
- |
| AUA1: Addis Ababa University Alternaria isolate 1, AUF5: Addis Ababa University Fusarium isolate 5, ++++: Very high antagonistic activity (>75 PIRG), +++: High antagonistic activity (61-75 PIRG), ++: Moderate antagonistic activity (51-60 PIRG), +: Low antagonistic activity (<50 PIRG), -: No antagonistic activity, Different alphabets depicted in superscript in the columns indicate mean treatments that are significantly different according to Tukey's HSD posthoc test at p<0.05, Each value is an average of 9 replicate samples±Standard error | |||||||
Effect of volatile metabolites: The volatile compounds released by Trichoderma species have also exerted inhibitory effect on the growth of the test pathogens. The T. asperelloides (T-97 and T-131) showed highest inhibition effect on the percent mycelia growth of both AUA1 (92.16-93.65%) and AUF5 (92.2-93.65%) (p<0.05), respectively. Least mycelium growth inhibitions (%) of AUA1 and AUF5 were occurred in the interaction with T. viride isolates T-158 (44.17%) and T-158 (44.2%), respectively. Most of the species showed percent mycelium inhibition values ranged between 65.8-77.9% against the test pathogens. The remaining species were poor producers of diffusible metabolites. The interaction between test pathogens and Trichoderma isolates were determined and illustrated as shown in Table 4.
Effect of non-volatile/diffusible metabolites of bio agents: In this experiment, results demonstrated significant differences in inhibitory effects of the non-volatile metabolites obtained from the different Trichoderma species. In the present study, the most efficient Trichoderma isolates overall was both T. asperellum isolate T-11 and T-12, which inhibited the two phytopathogenic fungal isolates (AUA1 and AUF5) growth by diffusible assays (90.12%) significantly (p<0.05). The remaining Trichoderma isolates displayed a range of antagonist activity from 16.38-88.77% (Table 4).
DISCUSSION
This study clearly demonstrated that Alternaria and Fusarium species are present in a high proportion of sesame seeds collected from various regions in Wolkait district, Northern Tigray that suggested the existence of inoculum to initiate disease outbreaks under favorable conditions to pathogen growth and development. Species delineation within the genus Alternaria and Fusarium requires careful attention in order to determine the range of isolates causing diseases on a sesame host. Among the major diseases that affect sesame is Fusarium wilt and Alternaria leaf blight, which are caused by Fusarium oxysporum sesami and Alternaria sesami, respectively47. In the present study, 12 fungal isolates were isolated, among them 5 of the isolates of Alternaria, 6 isolates of Fusarium and Cercospora were recorded from sesame seeds, leaves and soils collected from Wolkait district, Northern Tigray (Table 2). It was observed that the majority of isolates were obtained from Lalay Mayhumer followed by Bet-Mulu and Kalema. Similarly, Shekharappa and Patil48 has observed symptoms on infected plants included lesions with concentric rings on leaves that often coalesce to form large blighted leaf areas.
On the other hand, Fusarium wilts caused by Fusarium oxysporum f. spp. Sesami (FOS) is a devastating disease infecting sesame crop right from seedling to maturity resulting in crop losses in varying degrees depending on the severity of infection. It has been reported as the most important soil borne fungal disease in which the water-conducting (xylem) vessels become blocked, so that the plant wilts and finally dies48. The symptoms of leaf blight found on the leaves were initially round to irregular, brown colored necrotic spots with concentric zonation demarcated with brown lines inside the spots on the upper surface. The symptoms of the disease in the present study were found to be similar to the typical symptoms of the disease described earlier by Belay49 and Jyothi et al.50. In severe infection, several spots would coalesce together by involving a major portion of the leaf blade and the affected leaves would dry and usually drop off. The disease was observed to occur at all stages of plant growth20.
In this study, seven Trichoderma isolates gave early germination as well as highest germination percentage. Strong antagonism by Trichoderma species against a range of soil borne plant pathogens has been reported by Patil et al.51. The present study determined the potential antagonistic variation of isolates of Trichoderma in the two-soil borne and foliar phytopathogens of Fusarium and Alternaria isolates. The antagonistic capabilities of Trichoderma species were assessed by the inhibition of Fusarium species and Alternaria species growth through the dual culture test and about 28.6% of the isolates showed the highest inhibition values significantly (p<0.05) greater than 75% (very high antagonistic), the rest of them showed high inhibition values in the range of 66.26-75%29. The results revealed that T. asperelloides T-131 was found to effectively inhibit the radial mycelial growth of the pathogen (79.49%) compared to all other isolates. Mulatu et al.40 has also reported that Trichoderma species against F. xylarioides did not show any clear zone but they overgrew the pathogen and occupied all over the media and totally surrounded the fungus in a test. Similar results were reported by Solanki et al.52 in their antagonistic study of Trichoderma species against Rhizoctonia solani. Kotasthane et al.53 in their antagonistic study of Trichoderma species against two phytopathogens (Sclerotium rolfsii and Rhizoctonia solani) under in vitro confrontation assay reported that T. viride isolate T14 showed 100% inhibition against R. solani. Akinbode et al.54 have shown antagonistic potentials of two Trichoderma species, in which T. pseudokoningii had better inhibition of the mycelia growth of Colletotrichum destructive than T. harzianum.
Trichoderma species were performed well as bio control agents when tested individually with Alternaria and Fusarium isolates on culture media. The in vitro evaluation of dual culture technique exhibited that the mycelial growth of the pathogenic fungal isolates was suppressed by the production of volatile and non-volatile compounds of Trichoderma isolates. Isolates, T. asperelloides T-97 and T-131 showed the highest inhibition effect on the percent mycelia growth of both AUA1 (92.16-93.65%) and AUF5 (92.2-93.65%) fungal diseases, respectively. Most of the isolates showed percent mycelium inhibition values between 65.8-77.9% against the test pathogens. The remaining isolates were poor producers of diffusible metabolites. Vey et al.55 have reported that there are large varieties of volatile secondary metabolites produced by Trichoderma species which play an important role in controlling the plant pathogens.
In the present study, the most efficient Trichoderma species overall were both T. asperellum, which inhibited the two phytopathogenic (AUA1 and AUF5) growth by diffusible assays (90.12%). The in vitro confrontation assay has shown to be a useful and reliable method for identifying the biocontrol efficacy of Trichoderma strains56. Trichoderma species have been reported to produce a plethora of secondary metabolites possessing antimicrobial activity57. The ability of Trichoderma species to control Fusarium wilt was reported before in tomato58. The results of this study are in accordance with previous results which mentioned the high capability of micro-organisms to control sesame wilt disease59 and agreed with previous studies that found application of Trichoderma species is very efficient to suppress Fusarium oxysporum sesami and can present an efficient method to control Fusarium wilt in sesame60. On the other hand, the results revealed that the antagonists significantly reduced the growth of Alternaria sesami either by competition (over growing) or by antibiosis (exhibiting inhibition zones). The inhibition of mycelial growth of Alternaria sesami by Trichoderma isolates could be obviously attributed to several possibilities of existence of microbial interactions such as; higher competitive ability, stimulation and antibiosis by these isolates over the test pathogen. The antagonism of Trichoderma species against many fungi is mainly due to production of acetaldehyde compound. Results of volatile assay revealed that volatile compounds present in the culture filtrate inhibited mycelial growth of pathogens effectively.
CONCLUSION
All Trichoderma isolates reported here may be considered as efficient biocontrol agents for Alternaria leaf spot and Fusarium wilt pathogens of sesame. The T. asperellum (T-11 and T-12) and T. asperelloides T-97 showed maximum biocontrol property against plant pathogens studied. These isolates showed consistent results in volatile and non-volatile activity under in vitro condition against both of the two fungal pathogen isolates tested. The highest mean inhibitory effect on the growth of the test pathogen isolates were achieved by T. asperelloides T-97 (76.42%) and T-131 (79.49%) against Alternaria species and Fusarium species, respectively, restricting it almost completely in plates as compared to the control consisting of any of the two test pathogens growing alone. Therefore, Trichoderma isolates significantly (p<0.05) inhibit the radial growth of the test plant pathogens studied. Further research on mass multiplication by large scale fermentation is proposed for greenhouse and field trials. Isolation and identification of antibiotics and their field level application in crop fields may prove to be advantageous.
SIGNIFICANCE STATEMENT
This study discovers Trichoderma species based biological control agents that can be beneficial for controlling Alternaria leaf blight and Fusarium wilt of Sesame. This study will help the researcher to uncover the great potential and demand for the incorporation of the fungal antagonist Trichoderma antagonist in crop protection. Thus, Trichoderma species were highly antagonistic towards test pathogens and potentially could be used in commercial agriculture to control sesame diseases.
ACKNOWLEDGMENT
This study was supported by Ethiopian Biotechnology Institute (EBTi), Ethiopia. The authors are gratefully acknowledging the Department of Microbial, Cellular and Molecular Biology of Addis Ababa University for the kind assistance in providing the laboratory facilities during the whole period of this research work.