alexa Evaluation of Arbuscular Mycorrhizal Fungi and Trichoderma Species for the Control of Onion White Rot (Sclerotium cepivorum Berk) | Open Access Journals
ISSN: 2157-7471
Journal of Plant Pathology & Microbiology
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Evaluation of Arbuscular Mycorrhizal Fungi and Trichoderma Species for the Control of Onion White Rot (Sclerotium cepivorum Berk)

Ararsa Leta and Thangavel Selvaraj*

Department of Plant Sciences and Horticulture, College of Agriculture and Veterinary Sciences, Ambo University, Ambo, P.O. Box 19, Ethiopia

*Corresponding Author:
Prof. Thangavel Selvaraj
Department of Plant Sciences and Horticulture
College of Agriculture and Veterinary Sciences
Ambo University, Ambo, P.O. Box 19, Ethiopia
Tel: +251-913073294
Fax: +251-112362037
E-mail: [email protected]

Received date: December 07, 2012; Accepted date: January 21, 2013; Published date: January 26, 2013

Citation: Leta A, Selvaraj T, (2013) Evaluation of Arbuscular Mycorrhizal Fungi and Trichoderma Species for the Control of Onion White Rot (Sclerotium cepivorum Berk). J Plant Pathol Microb 4:159. doi:10.4172/2157-7471.1000159

Copyright: © 2013 Leta A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

The present study was carried out to evaluate the indigenous Arbuscular Mycorrhizal Fungi (AMF) and
Trichoderma species isolated from rhizosphere soils of onion cultivated fields at Ambo and Toke Kutaye districts of West Showa, Ethiopia, and their effect on plant growth, and their biocontrol against onion white rot caused by Sclerotium cepivorum Berk. Five species with twenty isolates of Trichoderma were isolated and screened in vitro for the inhibition of S. cepivorum. Out of these, four isolates of Trichoderma spp viz. T. harzianum (ATh1), T. viride (ATv1), T. hamatum (NThm3), and T. koningii (QTk2), were found potent antagonists with mean percent inhibition of the pathogen, 65.4, 64.8, 54.3 and 53.5, respectively. Altogether, 10 AMF species representing four genera viz. Acaulospora, Gigaspora, Glomus, and Scutellospora, were isolated and identified. Six dominant species of AMF were selected and mass multiplied by using Sorghum vulgare Pers. as the compatible host plant. Among six AMF species, the potential efficient strain, Glomus aggregatum (Awaro isolate), was selected for using as bio control agent. The bio-control potential of these bio-agents against white rot pathogen was carried out under pot culture condition, using G. aggregatum alone or in combination with four isolates of Trichoderma spp. Incidence of S. cepivorum was significantly reduced in bulbs of onion (66.19%), and also improved plant growth was observed in plants inoculated with combined inoculation of G. aggregatun and T. harzianum (ATh1 isolate), followed by the combined inoculation of G. aggregatum and T. viride (ATv1) with pathogen (59.75%). Plants inoculated with S. cepivorum alone showed pronounced disease symptoms with mean disease incidence of 90.5%. The overall reduction in the incidence of white rot was 56.22% in the treatment of T. harzianum ATh1 isolate with pathogen, followed by 53.72% for G. aggregatum with pathogen. These results clearly pointed out that G. aggregatum and T. harzianum ATh1 isolate can
block the severity of disease caused by S. cepivorum in onion. Use of these bio-control agents could be promoted as an active component of bio-intensive Integrated Disease Management Program (IDMP), under organic mode.

Keywords

Arbuscular mycorrhizal fungi; Trichoderma spp.; Biocontrol; Onion; White rot; Sclerotium cepivorum

Introduction

Allium crops are the most indispensable vegetable crops used as condiments in most Ethiopian cuisine. Among them, onion (Allium cepa L.), rightly called as “queen of kitchen”, is one of the oldest known and an important bulbous vegetable crop grown in Ethiopia. It is used in preparation of different foods, and in therapeutic medicine in the country. Besides, it is rich in flavonoids like quercetin and sulfur compounds, such as allylpropyl disulphide, that have been perceived benefits to human health [1]. Onion has also medicinal value, as a possible cancer preventive [2,3]. The best growing altitude for onions under Ethiopian condition is between 700 and 1800 MASL (Meters Above Sea Level) [4]. A survey made in 1987 estimated the total area under onions and shallot in Ethiopia to be about 10,000 ha, with the total production of about 75-500 tones. But, now-a-days the area under the production of onion is increased by far and productivity also increased up to 80-120 kg/ha under optimum condition [4]. However, the productivity of onion is affected by many biotic and abiotic stresses, resulting in yield reduction, low quality and less storability of the crop, and other constraints in the production of onion in Ethiopia, including the use of low quality seeds, imbalanced fertilizers, and uneven irrigations.

Among the diseases of onion, white rot (Sclerotium cepivorum Berk.) is identified as the most important disease of onion in Ambo and Toke Kutaye districts of West Showa, Ethiopia, causes breaking of floral stalks, and thus, the bulb yield and seed production is significantly reduced. In Ethiopia, the management methods of the diseases of onion are followed as mainly crop rotation, avoidance and sanitation to proper storage methods [5]. Conventional methods of control of diseases of onion include the use of chemicals, organic means, and through integrated management efforts. As a result, attempts to manage the disease have focused on reducing the populations of sclerotia in the soil through biological control. Research on biological control of plant pathogens has Received date: much attention in recent years, as a means of increasing crop production by avoiding a number of problems related to chemical control, and hence, developing practices compatible with sustainable agriculture [6].

Arbuscular mycorrhizal fungi (AMF) are widespread in nature, and a fundamental component of agro ecosystems. AM symbioses may also improve plant health through a more specific increase in protection (improve resistance, and/or tolerance against biotic and abiotic stresses) [7,8]. Trichoderma spp. as a potential bio control agent was recognized in the early 1930’s [9]. Among different strategies for plant growth promotion and disease suppression, biological approaches are very useful, and an ominous choice. The use of AMF and an antagonistic fungus, Trichoderma spp. are the major players, which could play active role in plant growth promotion and disease suppressions. Attempts have been made to know the importance of white rot disease on some onion growing areas, but not much detailed studies on disease survey works have been carried out, particularly in Ambo and Toke Kutaye districts of West Showa, Ethiopia. Hence, the present study was carried out to evaluate the native strains of AMF and Trichoderma spp. isolated from rhizosphere soils of onion cultivated fields at Ambo and Toke Kutaye districts of West Showa, Ethiopia, and their effect on plant growth promotion, and biological control activities against white rot disease.

Materials and Methods

Description of the study areas

The screening experiment was conducted in Ambo and Toke Kutaye districts of West Showa zone, Oromia Regional State, Ethiopia, during the academic year, 2008–2009, to evaluate the indigenous AMF and Trichoderma species from rhizosphere soils of onion cultivated fields at Awaro and Qora from Ambo district, and Nagafile and Imala Dawe (I/D) Ajo from Toke Kutaye district of West Showa, Ethiopia. Ambo district has total geographical area of 83598.69 sq.kms, which is located at 8°57’ North latitude and 38°07’ East longitude, at an average elevation of 1380-3300 meter above the sea level, and the Toke Kutaye district has total geographical area of 78887 sq.kms and is located at 8°57’ North latitude and 38°07’ East longitude, at an average elevation of 1800-2300 meter above the sea level. In both districts, the annual rainfall ranged from 800-1000 mm and the temperature of the districts ranged between 15°C and 29°C, with average temperature of 22°C.

Sample collection

The soil and root samples were collected from four different onion cultivated field study sites. At each study site, an area of 500 sq. meters was chosen for sampling. Five healthy and diseased plants were selected, and their roots, bulbs and rhizosphere soil samples were collected at 0-30 cm soil depth. Approximately, 2 kg of rhizosphere soil was collected in triplicates from each study site, and the samples were brought to Ambo Plant Protection Research Center (APPRC) laboratory in sealed plastic bags, and stored at 5-10°C. The roots of the healthy test plants were separately washed thoroughly, free of attached soil particles, and cut in to 1 cm bits and fixed in Formalin: Acetic acid: Alcohol (FAA) in the field itself [10]. The diseased bulbs were collected from the field in sealed plastic bags and stored at 5–10°C, until processed for use. Soil samples were thoroughly mixed and a portion of soil samples were analyzed for soil texture, pH, EC, N, P, K, Ca, Mn and Fe [11]. The remaining soil samples were used to isolate AM fungal spore populations and Trichoderma species.

Identification and mass production of AMF

Spore population density and species richness of AMF from each soil sample was estimated by the method of wet sieving and decanting [12]. Assessment and the percentage of AMF colonization in the roots of onion were determined by the method of trypan blue in lactophenol [10]. For identification of AMF, intact spores were picked up from the filter paper and mounted on micro slides in lactophenol, and were observed under binocular research microscope. The morphology of spores and sporocarps of AMF were observed, and then their characters were used for identification by using Manual for identification of AM fungi [13]. Mass inoculum production of AMF was carried out in the roots of Sorghum vulgare Pers. under glass house condition, by the method of Selvaraj et al. [14]. The selected dominant six species of AMF viz., Acaulospora scrobiculata, Glomus aggregatum, G. fasciculatum, G. deserticola, Gigaspora margarita and Scutellospora heterogama collected from four different study sites were only mass multiplied. Among them, Glomus aggregatum was selected for the efficient strain for further experimental use.

Identification and mass multiplication of Trichoderma spp.

Trichoderma species were isolated from rhizosphere soils of four different onion cultivated fields of West Showa, Ethiopia, using standard serial dilution plating technique on Trichoderma Selective Agar (TSA) medium [15], and identified down to species level based on standard mycological methods, with the help of the Manual of Soil Fungi [16], and the manual of Hypomycetes and identification of Trichoderma species [17]. Mass multiplication of Trichoderma species was followed by the method of Gopinathan and Selvaraj [18].

In-vitro evaluation

The pathogen, S. cepivorum, was isolated from bulbs of infected onion plants grown in well developed sick plot at APPRC, Ambo, and showing typical symptoms of the disease [19]. The isolated pathogen and the Trichoderma species were maintained as pure cultures at 4°C in refrigerator, until used. Twenty isolates with five species of Trichoderma were evaluated in-vitro for their antagonistic and inhibition potential against the onion white rot, using dual culture technique with direct confrontation test. Each of the twenty Trichoderma isolates and S. cepivorum were inoculated on to PDA (Potato Dextrose Agar) medium separately, and incubated at 25°C for 5 days. Control plates of the pathogen and the antagonist were also prepared. A Completely Randomized Design (CRD) was used. Isolates were then scored for degree of antagonism after five days, using the rating system of Bell et al. [20], on a scale of 1-5, where class1=Trichoderma completely overgrow the pathogen and cover the entire medium surface; class 2=Trichoderma overgrow at least 2/3 of the medium surface; class 3=Trichoderma and Sclerotium each colonize 50% of the medium surface, and neither of them appear to dominate the other; class 4=Sclerotium colonizes at least two-thirds of the medium surface, and appear to withstand encroachment by Trichoderma, and class 5=Sclerotium completely overgrow the entire medium surface. According to this rating system, a Trichoderma isolate is considered as antagonistic, if the mean score was less or equal to class 2 and not antagonist if the number was greater than class 2. Another set of experiment was prepared for inhibition and colony growth tests. To determine the inhibition percentage of the pathogen by each of the tested antagonists, growth of S. cepivorum was recorded by measuring the diameter of the colonies. Percentage inhibition (I%) of its colony growth was then calculated using the following formula used by Whipps [21].

image

Where I(%) represents the average inhibition percentage; Treated indicates the average colony diameter of S. cepivorum in the presence of the antagonist, and Control is the average colony diameter of S. cepivorum without the antagonist.

Efficacy test in vivo

The experiment was conducted under glass house condition (12–15°C minimum and 26–30°C maximum temperature), at APPRC, Ambo, Ethiopia. The potential native AMF, G. aggregatum (Awaro isolate) and four species of Trichoderma isolates were evaluated for the biological control of white rot disease in onion, under pot culture condition [22]. Sterilized sand, decomposed animal dung, and sterilized sandy clay loam soil (1:1:2 ratio) (5 kg per pot) was used. Sclerotia of S. cepivorum was thoroughly mixed with surface soil (100 sclerotia per pot) and wheat bran: sand cultures of Trichoderma spp. (1 g/100 g soil) and AMF inoculum (50g/pot) were added to each pot, and thoroughly mixed with surface soil. Two onion seedlings (Variety Adama red) were planted in each pot. The experiment was laid out in Completely Randomized Design (CRD), with four replications and 12 treatments consisting of various combinations of G. aggregatum, and different isolates of Trichoderma spp. with S. cepivorum were maintained in pot culture. Uninoculated and inoculated with S. cepivorum alone were used as control check.

The following treatments were:

• Control (without Sclerotium cepivorum, AMF, and Trichoderma spp.)

• Control (Sclerotium cepivorum (Sc) alone),

Gloms aggregatumu (Ga) alone

• Sc+Ga

• Sc+T. harzianum ATh1 isolate

• Sc+T. koningii QTk2 isolate

• Sc+T. hamatum NThm3 isolate

• Sc+T. viride ATv1 isolate

• Sc+Ga+ATh1 isolate

• Sc+Ga+QTk2 isolate

• Sc+Ga+NThm3 isolate

• Sc+Ga+ATv1 isolate

Data analysis

After 90 days of inoculation, the plants were uprooted and the data were recorded on plant height of healthy and diseased plants, shoot, root, and bulb dry biomass of plants, after drying the samples at 60°C to constant weight in a hot air oven. AMF root colonization and spore numbers in root zone soils were assessed by grid line intersection [10], and wet sieving and decantation methods [12], respectively. The per cent white rot infection was calculated using the following formula:

Per cent white rot infection=Total No. of diseased plants/Total No. of seedlings planted×100

The data were subjected to analysis of variance, and the treatment means were further separated by Duncan’s Multiple Range Test (DMRT), for significant differences at the level of P>0.05%.

Results and Discussion

Edaphic characteristics

Edaphic characteristics of the soils of the study areas indicated that the changes in soil moisture ranged from 9.2-10.5%, were in accordance with the climatic changes during different seasons, and the soil pH was acidic (pH 6.4–6.8) in all the four study sites, with low to moderate electrical conductivity (EC 1.4–1.8). The soil types were black sandy clay loam in Awaro, Qora and Nagafile sites, whereas dark grey clay loam in I/D Ajo. Generally, the soils were nutrient deficient, particularly high organic carbon (1.95–2.95%), less P-level (2.2–2.8 mg/ kg), moderate to high level of K (296–346 mg/kg), and also with other micronutrient content such as zinc (1.4–2.2 g/g), calcium (1.2–1.8 g/g), manganese (2.8–3.4 g/g), and iron (58.5–98.5 g/g). The available N content of the soils, irrespective of the study sites, were invariably high (625-860 mg/kg).

AMF colonization

The test plant was positive for AMF colonization in the roots of onion, although the species of AMF colonizing the organism was varied. Percent colonization of AMF in the roots of onion differed in each locality. But there was a definite trend in AMF colonization between the different characteristics of the soil. There was a certain degree of specificity among the different AMF species in those four study sites (Table 1). The presence of high degree of AM colonization with various AMF structures, such as infection pegs, hyphal coils (pelotons), hyphal dimorphism, intracellular arbuscules, inter and intracellular vesicles, were observed in the root cortical cells of onion. Rajeshkumar and Selvaraj [23] kept in mind while describing a species as mycorrhizal, if roots contained one of the following combinations of AM fungal structures in the primary cortex, hyphae+arbuscules, hyphae+pelotons, or hyphae+vesicles. Totally, ten AM fungal species were isolated from rhizosphere soils of the onion (Table 2). Of the ten AMF, only two species, G. aggregatum and G. fasciculatum, were found to be colonized in the roots of onion. The difference in colonization could be due to variation in the species and the soil type, upholding the view expressed by Schenck and Kinlock [24].

Location Percent AMF root colonization Total No. of AMF spores/100 g of soil Positive for AMF in the roots Positive for AMF in the root zone soils
S1-Awaro 96.5 a 1085 a Glomus aggregatum Glomus aggregatum, G. deserticola, Acaulospora delegata, A. scrobiculata, Gigaspora margarita.
S2-Qora 84.5 b 986 b Glomus aggregatum Glomus aggregatum, G. fasciculatum, Acaulospora scrobiculata, G. margarita
S3-Nagafile 56.5 c 425 c Glomus fasciculatum G.aggregatum, G. fasciculatum, G. geosporum, G. macrocarpum, G. margarita, Scutellospora heterogama.
S4-I/D Ajo 32.6 d 154 d Glomus fasciculatum Glomus aggregatum, G. fasciculatum, G. margarita, S. calospora, S. heterogama.

Table 1: Colonization and spore density of AMF from rhizosphere soils of onion.

List of AMF S1* S2 S3 S4 Species frequency %
Acaulospora delegata + -- -- -- 25
A. scrobiculata + + -- -- 50
Gigaspora margarita + + + + 100
Glomus aggregatum + + + + 100
G. deserticola + + _ _ 50
G. fasciculatum -- + + + 75
G. geosporum -- -- + -- 25
G. macrocarpum _ -- + -- 25
Scutellospora calospora -- -- -- + 25
S. heterogama -- -- + + 50

Table 2: Occurrence of AMF from rhizosphere soils of onion.

Spores and sporocarps of AMF

Altogether, ten AMF species representing four genera viz., Acaulospora, Gigaspora, Glomus, and Scutellospora were recorded (Table 2), of which Glomus was the dominant genus. Based on the frequency of occurrence, the AMF species were identified and grouped as dominant (above 50%), and common (blow 50%) forms. Accordingly, Gigaspora margarita. Glomus aggregatum, G. deserticola, G. fasciculatum, Acaulospora scrobiculata and Scutellospora heterogama, were dominant forms, whereas A. delegata, G. geosporum, G. macrocarpum and S. calospora were constitute common forms (Table 2). The variation in spore population in onion also might be due to soil types. Higher number of AMF species was observed in black sandy clay loam soil study sites. The mean spore number per 100 g of soil varied from 365–1085 in root zone soils of onion. The lowest number of spores was recorded in root zone soil of onion (365/100 g) at I/D Ajo study site, whereas the highest number (1085/100 g) was observed in Awaro study site (Table 2). The AMF spore abundance was reported to be determined by the host plant species and environmental variables, than by AMF species [25]. Edaphic characteristics such as soil type [26], soil depth, soil pH [27], and soil fertility [28], were reported to influence AMF sporulation. The present study clearly highlights the soil edaphic factors that favor root colonization and sporulation of AMF, associated with onion crop. Rajeshkumar and Selvaraj [23] studied that AMF distribution is dependent on the host plant and certain ecological factors, such as organic carbon, soil pH, soil nutrients, and soil fertility. Although AMF is not host specific, they exhibit certain host preferences [29]. Also, the root colonization patterns may regulated by the host. McGonigle and Fitter [30] reported from the study of two native grasses and forbs in England, that AMF in the field showed a degree of ecological specificity.

Mass production of AMF

Plants inoculated with G. aggregatum had significantly higher percent root colonization, extra-matrical spore count, shoot and root length, and shoot and root biomass (Table 3). Gigaspora margarita and G. fasciculatum were found to be the second best, next to G. aggregatum. All the parameters were found to be significantly least in plants inoculated with Scutellospora heterogama and A. scrobiculata.

Inoculation treatment Plant height (Shoot and root length) (cm) Plant ( Shoot and root) biomass (g/plant ) % AMF root colonization No. of AMF spores/ 100 g soil
Un inoculated control 85.0 d 24.90 d 0.00 e 0.00 e
Acaulospora scrobiculata 94.1 c 26.68 c 62.6 d 486.00 c
Glomus aggregatum 106.4 a 35.53 a 94.8 a 878.00 a
Glomus fasciculatum 98.9 b 30.88 b 72.2 b 568.00 b
Glomus deserticola 96.8 b 29.46 b 68.4 b 493.00 b
Gigaspora margarita 100.8 b 32.69 b 78.4 b 596.00 b
Scutellospora heterogama 89.7 c 26.99 c 58.5 c 345.00 d
SEM 2.8 1.4 2.8 14.6
CD (P<0.05) 1.2 0.8 1.2 7.4

Table 3: Effect of AMF on plant growth of S. vulgare.

Isolation of S. cepivorum from diseased bulbs

Fluffy white cottony growth of the fungal mycelium was noticed on the surface of the medium. As the growth of the mycelium progresses, the mycelium becomes more compacted with numerous small spherical black bodies (Sclerotia), forming on this mycelial mat, which are approximately the size of a poppy seed or pinhead.

Mass multiplication of Trichoderma spp.

Twenty isolates of Trichoderma spp. were isolated from rhizosphere soil samples of Ambo and Toke Kutaye districts study sites. Isolates were numbered based on substrate from which they were isolated (Table 4). The five species of Trichoderma viz., T. harzianum, T. viride, T. virens, T. koningii and T. hamatum were identified. The species identities of all the isolates are still under determination. The isolates were mass multiplied on wheat bran and sand substrate. As reported by Singh et al. [31], wheat bran as the good substrate used for multiplication of the Trichoderma antagonists.

Test organisms Mean colony growth of S. cepivorum (cm) and Trichoderma species Mean % inhibition on S.cepivorum
T. harzianum
     ATh1
     QTh2
     NTh3
     ITh4
Sclerotium cepivorum(Control)

2.00 d
2.10 d
2.15 d
2.25 d
4.45 a

65.4 a
64.8 a
61.4 a
60.8 a
----
T. viride
     ATv1
     QTv2
     NTv3
     ITv4
Sclerotium cepivorum (Control)

2.10 d
2.35 c
2.43 c
2.54 b
4.45 a

64.8 a
54.3 b
48.6 c
43.6 c
----
T. hamatum
     AThm1
     QThm2
     NThm3
     IThm4
Sclerotium cepivorum(Control)

2.34 c
2.43 c
2.48 c
2.56 b
4.45 a

54.3 b
48.6 c
45.4 c
43.5 c
----
T. koningii
     ATk1
     QTk2
     NTk3
     ITk4
Sclerotium cepivorum (Control)

2.38 c
2.39 c
2.56 b
2.45 c
4.45 a

53.5 b
53.2 b
43.5 c
48.5 c
---
T. virens
     ATvn1
     QTvn2
     NTvn3
     ITvn4
Sclerotium cepivorum (Control)

3.15 a
3.24 a
3.45 a
3.23 a
4.45 a

35.6 d
33.8 d
32.5 d
33.5 d
-----
C.V. 7.66 12.4
LSD (0.01) 0.48 8.2
SE 0.14 2.2

Table 4: Interaction between S. cepivorum and Trichoderma species under in vitro.

In vitro evaluation

The mean colony growth of S. cepivorum in plates containing Trichoderma isolates ranged from 2.0 cm (T. harzianum) to 3.45 cm (T. virens). The antagonism test revealed that four of the twenty isolates which were scored less than 2 were highly antagonistic to the white rot pathogen. However, T. virens, which exhibited a score of 3.15-3.45, was not antagonistic at all. This isolate grew only up to 1 cm in 5 days, indicating that it was not antagonistic, whereas all the other isolates colonized and covered the colonies of S. cepivorum on the respective plates. This isolate was, therefore, not tested further. In contrast, its colony growth reached 4.45 cm after 7 days in control plates. The results of the data recorded 5-7 days after inoculation showed that there was a significant difference among the Trichoderma species, in suppressing the colony growth of the pathogen and inhibition percentage (Table 4). T. harzianum ATh1 isolate covered the plate completely on the 5th day incubation, while the other species grew and cover the plate on the 6th day of incubation. Meanwhile, in the control treatment, S. cepivorum grown, covered the plates on the 7th day of inoculation and producing sclerotia after the 10th day. Therefore, S. cepivorum did not grow at all when inoculated after 48 hours inoculation of Trichoderma species. Trichoderma isolates are known to rapidly colonize medium surface and substrates [32]. Trichoderma harzianum inhibited the colony growth of S. cepivorum by 65.4%, followed by T. viride (64.8%), T. hamatum (54.3%), and T. koningii (53.5%, which was also significantly different from each other. Akrami et al. [33] also found that T. asperillum and T. harzianum reduced 43.2% and 51.5% reduction in disease incidence, respectively. Up to 36% reduction of colony growth of F. oxysporum was obtained by using Trichoderma harzianum isolate [34]. In a similar study done by using culture filtrates of T. viride, Tesfaye [35] found that F. solani could grow only for 1.5 mm in 96 hrs (4 days). Alemu and Kapoor [36] also found that T. viride and T. harzianum inhibited the fungus, Botrytis gladiolorium that causes corm rot on Gladiolus. Antagonistic interactions of Trichoderma species with other fungi and mechanisms involved in the bio-control process are based on antibiosis, parasitism, induced resistance and competition [37], and also produced enzymes that have been proved to be involved in the antagonistic activity [38]. However, antagonistic fungi are specific in their antagonistic activity against specific fungi [39]. In vitro tests are suitable for selecting antagonistic organisms with a particular mode of action, but are very poor predictors of the activity of the organisms in the field [40]. According to Mpika et al. [41], efficiency of Trichoderma species in antagonizing plant pathogens is closely linked with local conditions. Arya and Kaushik [42] reported maximum inhibition of Fusarium oxysporum and Rhizotonia solani (74.7-75.2%), with Gliocladium virens, followed by T. harzianum (65.9-72.4%). Kapoor [43] reported that the maximum inhibition growth by T. harzianum against R. solani, Pythium debayanum, Sclerotinia minor and Fusarium oxysporum f.sp .pisi. In the present study also, T. harzianum ATh1 isolate showed maximum inhibition of S. cepivorum (65.4%).

In vivo evaluation

Inoculation with Glomus aggregatum alone increased the plant growth parameters to the maximum, while less plant growth was observed in the plants inoculated with S. cepivorum alone. In all treatments, G. aggregatum either alone or in combination with isolates of Trichoderma spp. and pathogen, showed significant growth response and reduced the disease incidence, when compared to un inoculated and S. cepivorum alone control plants (Table 5). G. aggregatum combination with T. harzianum ATh1 isolate was significantly increased the growth, biomass, and reduced the disease incidence and severity of white rot disease in onion plants caused by S. cepivorum. The overall reduction in the incidence of white rot was 66.19% for G. aggregatum, with T. harzianum ATh1 isolate. The lowest shoot, root and bulb dry weights were recorded in plants inoculated with S. cepivorum alone. Singh et al. [31] reported that native species of Trichoderma was controlled red rot of sugarcane by the enzymatic action of the metabolites released by bio-agent. Some workers also showed that AMF can also be used as biological deterrent of soil borne diseases of vegetable crops [7,18]. In the present study, the wheat bran: sand formulation of T. harzianum ATh1 isolate combined with inoculum of G. aggregatum, significantly reduced the white rot of onion caused by S. cepivorum.

Treatments Average height of healthy plants (cm) Average height of diseased plants (cm) Shoot and root dry weight of healthy plants (g/plant) Average bulb weight of healthy plants Per cent root rot incidence and severity*
Control ( without inoculants) 62.0 b ---- 6.98 b 12.4 b ----
Control check (Sclerotium cepivorum (Sc) alone) 24.5 a 12.5 a 1.24 a 6.8 a 90.5 (50.84) a
Glomus aggregatum (Ga) alone 68.8 e ---- 9.88 e 20.8 e ----
Sc+Ga 64.4 d 46.5 c 8.76 d 17.6 d 46.28 (21.62)c
Sc+Trichoderma harzianum (ATh1 isolate) 68.2 e 48.8 c 9.85 e 18.2 d 43.78(13.97)c
Sc+T. koningii (QTk2 isolate) 60.5 c 34.7 b 7.82 c 16.5 c 45.03(23.11)c
Sc+T. hamatum (NThm3 isolate) 59.6 c 32.6 b 7.45 c 16.4 c 56.25(35.74)b
Sc+T. viride (ATv1 isolate) 66.5 e 46.8 c 8.45 d 17.8 d 39..65(14.68)d
Sc+Ga+ATh1 isolate 69.5 e 52.4 d 9.95 e 21.2 f 33.81(11.31)e
Sc+Ga+QTk2 isolate 66.8 e 49.6 d 8.24 d 20.3 e 45.03(19.5)c
Sc+Ga+NThm3 isolate 64.5 d 40.6 c 8.75 d 19.8 e 56.28(23.11)b
Sc+Ga+ATv1 isolate 67.5 e 50.8 d 8.65 d 20.6 e 40.25(20.56)d
LSD 0.01 8.6 6.8 1.4 3.4 8.6
CD (%) 2.4 3.4 0.4 0.8 2.4

Table 5: Effect of Glomus aggregatum and Trichoderma spp. on growth and the incidence of onion white rot.

Conclusion

From the results of this study, it is concluded that the tested indigenous Trichoderma isolates have high potential to inhibit the colony growth of S. cepivorum. The efficacy test results of AMF and Trichoderma spp. clearly indicates that AM fungal inoculation alone, or in combination with Trichoderma spp. reduced the incidence and severity of white rot, and increased the plant growth of onion. Also, these two bio-control agents commercially exploited as bio-fungicides, and could be recommended to the farmers in Western Showa, Ethiopia for the management of white rot in onion. The outcome of bio-fungicides (Trichoderma and AMF) distribution under the lab to land program has clearly brought out the role of AMF and Trichoderma spp. in the management of plant disease in field conditions, at Western Showa, Ethiopia, which merit commercialization.

Acknowledgements

I am greatly indebted to my advisor Prof. Thangavel Selvaraj for his invaluable professional help, initiatives and encouragement. This research work also could have been realized without the consistent support of the whole staff of Ambo Plant Protection Research Center (APPRC) and Department of Plant Sciences of Ambo University, Ambo, Ethiopia.

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