Effect of Trichoderma asperellum and Trichoderma virens on Allium cepa L. Growth, Damping off and Basal Rot Disease Incidence and Severity in Sri Lanka

Trichoderma species are frequently used for the biological control of phytopathogenic fungi and they have also been reported as plant growth promoters. In the present study, the effect of two Trichoderma spp. i.e. Trichoderma asperellum and Trichoderma virens isolated from the soils of onion fields on the growth of Allium cepa L. plants and suppression of damping off and basal rot diseases was evaluated under field conditions. The two Trichoderma spp. were mass cultured in a low cost medium containing molasses and yeast and added to a low cost carrier medium consisting of talc. Two formulations, i.e. T. asperellum only and T. asperellum in combination with T. virens were prepared and the formulations were tested for their effect on onion seedlings at the nursery stage and also on transplanted plants in the field. At the nursery stage, the two formulations were applied using two methods i.e. soil application prior to planting of onion seeds or priming of onion seeds with the two formulations separately before planting. Both methods reduced the incidence and severity of damping off disease while increasing the growth of seedlings significantly (p ≤ 0.05) at the nursery stage. Additional treatment with the two formulations as seedling root dips or soil applications before transplanting the seedlings in the field were effective in controlling basal rot disease of A. cepa L. and enhancing the growth of Allium cepa L. plants significantly (p ≤ 0.05) in the field.


Introduction
Big onion (Allium cepa L.) is a condiment grown for its flavorful bulbs in Sri Lanka as well as a number of other countries in the world. In Sri Lanka about 627 Hactares is cultivated in the Matale and Anuradhapura districts. However, the local cultivation does not meet the annual requirement of big onion approximately 203,993 MT per year [1]. One major factor contributing towards yield reduction is infectious diseases that occur both during the nursery stage and in transplanted field conditions. Some of the more economically significant diseases that reduce yields are caused by fungal pathogens. Diseases caused by fungal pathogens can be broadly divided into seedling diseases, foliage diseases and bulb diseases. The most common fungal genera responsible for big onion diseases are Fusarium, Sclerotium, Pythium, Rhizoctonia, Colletotrichum and Alternaria [2,3].
Damping off disease of Allium cepa L. that infect young seedlings at the nursery stage is one of the most important diseases caused by soil-borne fungal spp. Fusarium, Pythium and Rhizoctonia either singly or in combination causing seedling mortality before they are transplanted [3]. This disease may manifest before or after emergence of seedlings i.e. pre-emergence or post emergence damping off.
Further, about 10-50% yield loss is caused by basal rot disease, in the transplanted fields and during storage [4]. Basal rot of Allium cepa L. is caused by the soil borne fungus Fusarium oxysporum f. sp. cepa and the symptoms are yellowing or necrosis and withering of the leaves, stunted growth and rotten watery bulbs with decaying root systems.
Chemicals are used extensively for the control of damping off and basal rot pathogens both at nursery stage and the transplanted crop and the more commonly used fungicides in Sri Lanka are Captan, Homai, Mancozeb, Thiram, Brassicol, Benlate, Cresent, Topsin M,Carbendazim [5,6]. However, the use of chemicals results in deleterious effects on soil organisms and adverse effects on the environment and human health specially as onions are sometimes consumed without cooking [7]. Therefore, safe alternatives should be sought to overcome these problems and the use of microorganisms with the ability to control pathogens has been reported as a safe and viable method. Microorganisms with Biological control ability have been used successfully to control a number of diseases in numerous crops and could be the best alternative especially against soil borne pathogens such as Fusarium spp. [8][9][10]. Trichoderma spp. have been used worldwide as effective bio control agents of many fungal pathogens i.e. F. proliferatum, F. solani, Sclerotium cepivorum [11]. However, exotic microorganisms which could be potentially damaged the native ecosystems and might feed on non-target hosts. Therefore, the present study aimed at isolating Trichoderma spp. occurring naturally in the soils of onion fields in Sri Lanka and testing their effectiveness in controlling the causative agents of damping off and basal rot diseases of onion with a view to utilize them as effective, safe and viable control agents of the two diseases under field conditions. For the preparation of a formulation to be applied under field conditions, the effective Trichoderma isolates need to be mass cultured. For the formulations to be economically viable, the mass culture medium must be cheap and easily accessible but at the same time should support a high level of sporulation and growth of the selected Trichoderma spp. Rotten Grains, Sugarcane bagasse, vegetable waste, fruit juice waste, Potato dextrose broth, Maltose peptone broth, rotten wheat have been reported as effective mass culture media in a number of studies carried out previously [12,13] and in the present study, a suitable mass culture medium was developed. A suitable carrier medium has to be selected when applying the mass cultured inoculum/inocula to the field, and a carrier medium should be sufficiently inert to discourage the growth of soil organisms but at the same time retain the viability of the inoculum. A suitable carrier medium that fulfills these requirements was also selected in the present study.
In addition to control of pathogenic fungi, Trichoderma spp. have also been reported to enhance growth of plants [14,15]. Therefore, the effect of the Trichoderma formulations on the growth of onion seedlings and plants was also evaluated under field conditions. This is the first report of preparing locally isolated Trichoderma virens and Trichoderma asperellum formulations for the effective control of the causative agents of damping off and basal rot diseases prevalent in commercial onion cultivations in Sri Lanka.

Biocontrol Agents and Pathogen Isolate
Trichoderma spp. used in this study were previously isolated using the method described by [16] from soil collected from onion fields in Sri Lanka [17] and Fusarium sp. was isolated from the A. cepa L. seedlings showing characteristic symptoms of damping off disease [17].
The Trichoderma isolates were identified as T. asperellum and T. virens. The Fusarium isolate was identified as Fusarium solani (NCBI GeneBank accession numbers obtained after deposition of accessions Trichoderma asperellum, Trichoderma virens and Fusarium solani are MG198706, MG199587 and MF685335 respectively) [18].

Method of Preparation of Trichoderma spp. Formulations
Formulation I The mass culture medium comprises a mixture of molasses and yeast. 500 mL of the medium was autoclaved (121 o C for 20 minutes) and then inoculated with 1 cm diameter eight agar discs of pure culture of Trichoderma asperellum only and incubated at room temperature while shaking at 180 rpm for 14 days.
Formulation II Four discs each of 1 cm diameter were cut from each Trichoderma spp. (i.e. Trichoderma asperellum and Trichoderma virens) and inoculated together into 500 mL molasses and yeast broth and incubated as mentioned above.
Both mass cultured preparations were added to autoclaved talc which served as the carrier medium at a 1:2 (v/w) ratio under aseptic conditions [19,20].
The field experiment conducted to test the effectiveness of the two formulations against damping off disease caused by Fusarium solani was extended up to transplanted stage of onion crop to test their effectiveness against the basal rot disease in the field.
Field experiments to test for the effect of Trichoderma spp. on Allium cepa L. diseases and growth Tests were carried out during nursery stage of cultivation of the crop in the field. L. Growth, Damping off and Basal Rot Disease Incidence and Severity in Sri Lanka

Preparation of Seed Beds-Nursery Stage
Field experiment using the seedlings at the nursery stage was conducted in a farmer nursery in Galewela during 17 th May-17 th June 2015 (yala season). Three to four weeks before planting the seeds, the land was ploughed and the soil turned to a 20-25 cm depth several times and left exposed to direct sunlight.
Three standard nursery beds of 3.6 m x 0.9 m x 0.15 m were prepared. Each bed was considered as a block ( Figure  1). Each block was divided into 12 plots of 0.3 m Χ 0.9 m Χ 0.15 m. Decomposed organic manure was incorporated into a 10 cm depth (10-15 kg/standard bed). Soil was turned once a week about 3 times and rice straw and rice husk was burnt on the beds. Rows 10 cm apart from each other were marked in each plot. Then a groove of about 12 mm depth was made along each row.
6.5 g of seeds of the onion cultivar Galewela light red were planted in each plot by spreading evenly in each groove. Seeds were then covered with a thin layer of soil. The seedbeds were then covered with light mulch and irrigated twice a week. After germination, of seeds, the mulch was removed. Hand weeding and watering was done until the seedlings are 6-8 weeks old when they were ready for transplanting.
Treatments were allocated to each plot in 3 replicate blocks and the distribution of treatments was as indicated in Figure 1. Twelve treatments tested in the trial (Table 1) were distributed as a Randomized Complete Block Design (RCBD), in the three replicate blocks.
During the field trials, soil populations of Trichoderma spp. was monitored using serial dilution tests.
The details of treatments and the quantities used are as depicted in Table 1. Table 1. Treatments tested in the nursery field trial. Trichoderma asperellum in talc (Formulation I) was applied to batches of seed 6.5 g in 50 mL conical flasks. The flasks were gently rotated for 10 min. to distribute the powder homogenously (as seed treatment) + artificial soil inoculation with Fusarium solani C.C.M. was applied as a 75-mm layer

T4
Trichoderma virens and Trichoderma asperellum together in talc (Formulation II) was applied to batches of seed 6.5 g in 50 mL conical flasks. The flasks were gently rotated for 10 min. to distribute the powder homogenously (as seed treatment) + artificial soil inoculation with Fusarium solani C.C.M. was applied as a 75-mm layer T5 Standard fungicide treatments were done according to the recommendations given by the Department of agriculture. The seeds were treated with Thiram at 2 g/kg of seed before sowing, The top soil of nursery was treated with combination of Thiram 80%, Thiophanate methyl 70% WP and nursery was drenched with the same chemical at 2 g/ liter of water at fortnight interval + artificial soil inoculation with Fusarium solani C.C.M. was applied as a 75-mm layer Trichoderma asperellum in talc (Formulation I) was applied to batches of seed 6.5 g in 50 ml conical flasks. The flasks were gently rotated for 10 min. to distribute the powder homogenously (as seed treatment) + natural Fusarium solani inoculum present in the soil

T10
Trichoderma virens and Trichoderma asperellum together in talc (Formulation II) was applied to batches of seed 6.5 g in 50 ml conical flasks. The flasks were gently rotated for 10 min. to distribute the powder homogenously (as seed treatment)+ natural Fusarium solani inoculum present in the soil T11 Standard fungicide treatments were done according to the recommendations given by the Department of agriculture. The seeds were treated with Thiram at 2 g/kg of seed before sowing, The top soil of nursery was treated with combination of Thiram 80%, Thiophanate methyl 70% WP and nursery was drenched with the same chemical at 2 g/ liter of water at fortnight interval + natural Fusarium solani inoculum present in the soil
Fusarium solani was mixed with composted chicken manure and applied in the nursery as a saw dust/rice bran preparation to obtain 1% (w/w) inoculum level. CFU of Fusarium solani within saw dust/rice bran preparation=5.0x10 3 CFU/g

Determination of effect of different treatments (Table 1) on incidence and severity of damping-off disease
The incidence and severity of the damping off disease in the treated seedlings was determined 20 days after sowing. A scoring system was developed as follows with 0-3 severity scales. 0=no infection; 1=yellowing and wilting of leaves; 2=stunting of seedlings (seedling length ≤ 12.0 cm); 3=collapsed seedlings and/or completely dead seedlings. Fifty onion seedlings were sampled randomly from each plot. Disease incidence was calculated using the following formula Disease incidence ! 100 (1) The disease severity index (DSI) ranged from 0 (no disease) to 100 (all plants killed) and was calculated for each treatment by using the following formula: where class indicates 0-3 and the maximum disease grade indicates 3.

Determination of effect of Trichoderm formulations on the growth of plants
Plants were harvested from each plot and transported to the laboratory to obtain measurements.
The length of the starting point of stem to the end point of the flag of the longest leaf of Allium cepa L. seedlings was measured as the seedling length. The lengths of the longest root along with two other randomly selected fibrous roots were measured per onion seedling.

Statistical Analysis
The means were analyzed by analysis of variance (ANOVA) and Tukey's test at 5% significant level with Minitab 16 statistical software.

Preparation of Land for Transplanting
Field experiment using the Allium cepa L. plants at the field was conducted in a farmer field in Galewela during 17 th June 2015-05 th September 2015 (yala season). The 30 day old seedlings with 3-4 leaves, 18-20 cm height and with slightly marked bulbs were selected prior to transplanting. Welldrained land was selected. The land was ploughed two to three times to bring the soil to a fine tilth. Primary weed control was done 10-14 day prior to land preparation manually and by using selective weed killer oxyfluorfen (Goal). 6 m Χ 0.9 m standard size, flat, raised beds were prepared and there were six of such beds. The beds were soaked well by giving pre-transplanting irrigation using sprinkler system. Each of these standard beds was divided into two equal plots of 3 m Χ 0.9 m.
Seedlings grown under treatments T1-T12 mentioned in the previous nursery stage trials were uprooted and separately transplanted in the beds 10 cm distance from each other. 300 seedlings were uprooted from each treated plot and were replanted in the prepared beds in the field.
Eleven treatments were tested in the trial. Initial treatment within the nursery field trial and subsequent treatment within the transplanted field trial are given in the Table 2.
Field experiment to test the effect of Trichoderma spp. on basal rot disease and on the growth of onion plants The transplanted seedlings were treated as follows:

Effect of Treatments on Incidence and Severity of Basal Rot Disease of Allium cepa L.
The incidence and severity of the basal rot was determined nine weeks after transplanting. The infection was identified on basis of symptoms i.e. yellow and tan to brown leaves, necrosis of leaf blades, withering of leaves, discoloured and rotten watery bulbs. Disease incidence was calculated as number of infested plants showing anyone of the above mentioned symptoms out of total numbers (300) of A.cepa L. plants observed.
The disease severity index (DSI) ranging from 0 (no disease) to 100 (all plants killed) was calculated for each treatment by using the following formula: where class indicates 0-3 and the maximum disease grade indicates 3 The following disease classes were established for the assessment of disease severity
In order to evaluate these parameters, the A. cepa L. plants were harvested and transported to the laboratory to obtain measurements. The length of the starting point of stem to the end point of the flag leaf of transplanted A. cepa L. plant was measured as the shoot length. The lengths of the longest root along with two other randomly selected fibrous roots were measured per Allium cepa L. transplanted plants. Dry weight was measured by oven drying the plants at 60 o C until a constant weight is reached. Total fresh weight consisting leaves along with fibrous roots were measured as transplanted A. cepa L. plant fresh weight.

Statistical Analysis
The means were analyzed by analysis of variance (ANOVA) and Tukey's test at 5% significant level with Minitab 16 statistical software.

Results and Discussion
Big onion cultivations in Sri Lanka are mostly concentrated in the Matale and Anuradhapura regions yielding about 75,776 MT annually [21]. However, the amount harvested is insufficient to meet the local demand and one factor that contributes significantly towards reduced yields is diseases that affect onion cultivations deleteriously at different stages of growth.
The survey carried out in a previous study revealed that the more common diseases that affected the plants in all areas of cultivation in Sri Lanka included seedling damping off at the nursery stage and basal rot, leaf and flower stalk anthracnose (twister) under field conditions. The causative agents of these diseases were identified as Fusarium spp., Colletotrichum gloeosporioides, Alternaria sp. and Sclerotium sp. Amongst these diseases, damping off and basal rot were the more important production constraints. The pathogenicity of Fusarium solani as the causative agent of damping off disease of A. cepa L. was confirmed by following Koch's postulates [22,17].
Fungicides such as Thiram, Homai, Brassicol, Benlate, Captan, Cresent are used currently for the control of onion diseases, Use of agrochemicals has resulted in contamination of the water table and the soil environment resulting in deleterious effects on the environment. Many health problems including the CKDu prevalent in Sri Lanka are suspected to be due to the harmful effect of chemicals used in agricultural practices [23].Therefore, less harmful but effective means of disease management and increased crop productivity should be sought and the use of microorganisms as an alternative to chemicals is a fast developing concept. Microorganims have proven to be effective in the control of pathogens of numerous crops [8,9].
Trichoderma spp. that are common saprophytic fungi found in almost any environment including many diverse soils, have been used as biocontrol agents due to their ability to reduce the incidence of disease caused by plant pathogenic fungi by means of a wide range of mechanisms. [10,[24][25][26]. Successful control of soil borne plant pathogenic fungi such as Rhizoctonia, Sclerotium, Sclerotinia, Fusarium, Pythium, Phytophthora that cause diseases in onion as well as in other crops by Trichoderma spp. have been recorded in Sri Lanka and other countries of the world [27][28][29][30][31][32].
The ability of Trichoderma spp. to control plant diseases are attributed to a number of mechanisms such as competition, mycoparasitism, formation of restrictive structures, antibiosis, and production of secondary metabolites and even induction of resistance in the plant [14,33,34].
Introduction of exotic isolates to a soil environment can cause damage to the soil organisms and upset the ecosystem dynamics. Therefore, in the current study, the Trichoderma spp. that could be utilized as bio control agents of the pathogens of onion were isolated from local onion fields with a view to select the most effective candidates for the control of causative agents of damping off and basal rot diseases of onion as these two diseases cause considerable economic losses.
Two of the Trichoderma isolates i.e. Trichoderma virens and Trichoderma asperellum isolated from the onion fields in Sri Lanka showed high control ability of phytopathogenic fungi Fusarium sp., Colletotrichum gloeosporioides, Alternaria sp. under in vitro conditions by using mechanisms such as competition, mycoparasitism, formation of restrictive structures [35] and therefore the two spp. were selected for further tests under green house and field conditions. This is the first report of the two Trichoderma spp. being present in agricultural fields in Sri Lanka.
In order to prepare formulations of the effective bio control agents, a suitable multiplication medium should be developed. The medium should support a steady state of growth and sporulation of a fungal bio control agent and in the present study, Molasses was developed as a multiplication medium.
Two locally available, natural substrates i.e. a sawdust based medium and molasses yeast medium were evaluated as a suitable medium for culture and the molasses yeast medium was selected to be more suitable as it facilitated sufficient sporulation (10 10 spores/mL) and growth of both Trichoderma isolates tested. High numbers of mature chlamydospores were also produced in the molasses yeast medium which may allow prolonged shelf life of the final preparation. As molasses is a byproduct of the sugar industry, it is accessible locally at a fairly low cost which makes molasses yeast a suitable medium in many aspects [20].
Once the selected bio control agent is cultured in the required scale, it has to be prepared as a formulation to be applied in the field. For field applications, the potential bio control agent must be incooporated into a carrier medium with minimum nutrient levels as high nutrients in the carrier medium will enable the growth of other competitive fungal spp in the soil. However, the carrier medium should retain the viability of the bio control agent as well maintaining the required level necessary for control of pathogenic fungi. In the present study, Talc was tested and proved to be suitable as a carrier medium. [20].
Several methods have been recommended for application of bio-fungicides for the successful management of plant diseases by several workers. The most common application strategies are seed biopriming, seedling dip (suitable for the crops where transplanting is practiced), soil application and foliar spray [36]. [9] used coated seeds and soil treatment with different combinations of Trichoderma spp. to control Fusarium rot of lentil and found out both reduction of disease severity and growth enhancement. According to the investigation carried out by [37], onion basal rot caused by Fusarium oxysporum f.sp. cepa could be effectively controlled with soil amendment with Trichoderma spp.
Based on these reports, two methods i.e. seed coating and soil inoculation were tested for the introduction of the prepared Trichoderma inoculum under greenhouse conditions and the results showed that seed priming and soil treatment with Trichoderma spp. gave the best retention rates and bio control [38].
The field trials were carried out during the yala season in a farmer field in the Galewela area. The level of the inoculum in soil was monitored under field conditions and it increased from 10 cfu/g to10 4 cfu/g after 1 month and remained at a fairly high level of about (10 4 -10 5 cfu/g) upto 3 months and reapplications of the inoculum was considered to be unnecessary.

Effect of Treatments on Damping off Disease in Onion
Seedlings at the Nursery Stage DI and DS of A. cepa L. seedlings were evaluated 20 days after sowing (Table 3). The highest average of damping-off disease incidence on A.
cepa L. seedlings occurred in the T 6 (80.667%) when Fusarium solani was inoculated to the plots artificially while T 5 (Fusarium solani + standard fungicide application) recorded the lowest damping off disease incidence (8.00%). The indices of severity for untreated plots (T 12) and soil incorporated with Fusarium solani alone (T 6) were 18.222% and 47.667% respectively and these values were significantly (p ≤ 0.05) different from rest of the treatments (T 1-T 11) that had low disease severity indexes. Control of A. cepa L. seedling damping off with the effective Trichoderma spp. application as either seed coating or soil inoculation was comparable with control obtained with the fungicide treatment.

Determination of the Level of Trichoderma spp. Inoculum in Soil During the Field Trial
The level of the Trichoderma spp. inoculum in soil was monitored under field conditions and remained at 10 4 CFU/g after 1 month.

Effect of Different Treatments on Seedling Length (cm) of Allium cepa L. (20 Days, 30 Days After Sowing)
Treatments i.e. T 4 and T 9 had a significantly (p ≤ 0.05) larger seedling height (16.2533±0.33 cm, n=15 and 16.2933±0.12 cm, n=15 respectively) than other treatments at 20 days of seedling growth. The shortest seedlings were recorded in treatments having no fungicides and bio-agents i.e. 12.1467±0.27 cm, n=15 mean value for seedlings infected by Fusarium solani (T 6) and to 13.94±0.16 cm, n=15 mean value in the uninfected control seedlings (T 12).
Results revealed that the average of 30 days old A. cepa L. seedling height with Trichoderma spp. application were in the range of 23.8933±0.68 cm -26.92±0.58 cm, n=15 compared to 15.9067±0.28 cm, n=15 in the control seedlings infected by Fusarium solani and to 21.5±0.48 cm in the uninfected control seedlings. No significant differences were recorded among Trichoderma treatments and standard fungicide treatments, while significant differences were recorded between Trichoderma treatments and the Fusarium solani inoculated and Fusarium solani non-inoculated controls. Significantly (p ≤ 0.05) least seedling height was obtained in pots where Fusarium was inoculated alone (control) i.e. 15.9067±0.28 cm, n=15 followed by uninfected control seedlings (Table 4).

Effect of Different Treatments on Root Length (cm) of Allium cepa L. (30 Days After Sowing)
Seeds previously treated with both Trichoderma spp. (Trichoderma virens, Trichoderma asperellum) produced the longest fibrous roots. Means comparisons showed that there was significant difference between Trichoderma spp. inoculated treatments and the Fusarium solani inoculated and Fusarium solani non-inoculated controls (Table 4). L. Growth, Damping off and Basal Rot Disease Incidence and Severity in Sri Lanka

Effect of Different Treatments on the Number of A.cepa L. fibrous Raoots
The number of average roots per seedling as result of application of Trichoderma spp. were in the range of 9.2±0.73 -11.2±0.58 roots/seedling, n=5. No significant differences were recorded among Trichoderma spp. inoculated treatments, standard fungicide treatments and Fusarium solani non-inoculated control (T 12) while significant differences were recorded between Treatments 1,2, 3,4,5,7,8,9,10,11,12 and the Fusarium solani inoculated control (T 6) ( Table 4).

Effect of Different Treatments on Seedling Fresh
Weight of Allium cepa L. (30 Days After Sowing) By looking at fresh weight measurements, a significant difference (p ≤ 0.05) in fresh weight between all treatments was observed. Lowest mean seedling fresh weight (0.460667±0.04 g, n=15) was recorded by Fusarium solani inoculated control (T 6). Higher seedling fresh weight (1.19973±0.06 g, n=15) was recorded with Trichoderma spp. (Trichoderma virens, Trichoderma asperellum) + Fusarium solani inoculation (T 2) and were on par with rest of the Trichoderma spp. inoculated treatments i.e. T 4, T 7, T 9, T 10 ( Table 4).

Field Experiments to Test for the Effect of Trichoderma spp. on Basal Rot Disease of Allium cepa L.
The effect of each treatment on DI, DS and growth parameters of A. cepa L. plants and bulbs were evaluated at 9 weeks after transplanting.
In all cases DI and DS of A. cepa L. plants were evaluated at 9 weeks after transplanting. A. cepa L. basal rot disease incidence and disease severity levels were higher in the treatment TT 1 (neither composted chicken manure nor Trichoderma spp.) i.e. 83.33% and 51.00% followed by the treatment TT 2 (composted chicken manure without Trichoderma spp.) i.e. 79.33% and 45.22%. These A. cepa L. plants raised from the untreated nursery plots i.e. neither A. cepa L. seed treatment with Trichoderma spp. preparation nor soil inoculation using Trichoderma spp. preparation. Lower A. cepa L. basal rot disease incidence and severity indexes were recorded from rest of the treatments (TT 3-TT 11). Application of Trichoderma spp. i.e. either Trichoderma asperellum alone or in combination Trichoderma virens to the rhizosphere of A. cepa L. transplants decreased both disease incidence and severity (85.20% disease incidence reduction and 86.32% disease severity reduction relative to Trichoderma spp. non-inoculated controls). Pre-treatment of nurseries and subsequent treatment of seedlings before transplanting with Trichoderma spp. could be recommended for controlling both damping-off disease in the nurseries and basal rot disease of A. cepa L. in the field. Shoot growth of treatments TT 5, TT 6 and TT 11 was increased significantly compared to control treatments (TT 1 and TT 2). Mean shoot length of treatments TT 2, TT 3, TT 4, TT 7, TT 8, TT 9, TT 10 and TT 11 were statistically similar, but Trichoderma spp. based bio-fungicides multiplied in combination with composted chicken manure i.e. TT 3-TT10 were more effective treatments to increase shoot length as composted chicken manure provide a substrate for multiplication of Trichoderma spp.

Field Experiments to Test for the Effect of Trichoderma spp. on Growth of Allium cepa L.
The root growth of 10 day old A. cepa L. transplant was significantly enhanced by the different Trichoderma spp. based treatments as compared to the untreated controls. The range in root length was 2.7 -4.7 cm for treatments with Trichoderma spp. Number of roots per plant was also increased significantly compared to control treatments TT 1 and TT 2.
Treatment TT 10 significantly increased transplant fresh weight compared with the other treatments. The fungicide treatment (TT 11) mean fresh weight was comparable with the treatment TT 10.
The minimum fresh weight for 2 month old A. cepa L. transplants was recorded from TT 1 followed by TT 2. Application of bio-fungicides had significant effect on the A. cepa L. 2 month old plants compared with the untreated controls. Efficiency of all treatment strategies to increase fresh weight was statistically similar.
The mean maximum dry weight for 10 day old transplants was recorded in treatment TT 5 (Initial seed treatment with Trichoderma virens+ Trichoderma asperellum and subsequently seedling roots were treated with Trichoderma virens+ Trichoderma asperellum) followed by TT 11. All treatments with Trichoderma spp. based bio-fungicides had increased the dry weight and fresh weight. Both fungicide treatment (TT 11) and Trichoderma spp. inoculated treatments (TT 3, TT 4, TT 5, TT 6, TT 7, TT 8, TT 9, TT 10) significantly increased the weight of healthy bulbs compared with the untreated controls (TT 1 and TT 2). The maximum bulb weight was recorded in treatment TT 3 followed by TT 5.

Effect of Different Treatments on Allium cepa L. bulb
Diameter and Circumference Both fungicide treatment (TT 11) and application of Trichoderma spp. (TT 3, TT 4, TT 5, TT 6, TT 7, TT 8, TT 9, TT 10) significantly increased healthy bulb diameter and circumference (p ≤ 0.05) compared with the untreated controls. The healthy bulb diameter for Trichoderma spp. applied A. cepa L. planting was between 4.833 cm and 5.033 cm. The circumference for healthy bulb for Trichoderma spp. applied A. cepa L. planting was between 16.133 cm and 17.033 cm.
Results of the field trials showed that the inoculation of the Trichoderma spp. together and T. asperellum alone reduced post emergence damping off disease incidence and the disease level was significantly different (p ≤ 0.05) from that of the uninoculated controls. A significant disease reduction was evidenced even in the treatments where a high level of pathogen inoculum was added to the soil. However, inoculation of the two Trichoderma spp. in combination did not show an increased level of control of the disease when compared with T. asperellum inoculation alone. Similar inoculations of more than one Trichoderma spp are sometimes reported to be effective in controlling pathogens. By using a combination of T. harzianum, T. asperellum and T. virens it was possible to reduce disease incidence percentage of cucumber fields exposed to Fusarium pathogens such as F. solani and F. oxysporum as agents of root and stem rot cucumber under greenhouse conditions [39]. Similarly, sesame seeds treated with three isolates of Trichoderma viride reduced the pre and post emergence damping off caused by Rhizoctonia solani and Fusarium oxysporum f.sp. sesame under pot and field conditions. In the present study the level of control achieved by the Trichoderma inocula was not significantly different from that achieved by the fungicide treatment.
Among tested treatment strategies, initial seed treatment with Trichoderma spp. would be relatively more effective than soil infestation with Trichoderma spp. The amount of Trichoderma spp. added using soil application was much greater than that carried by the A. cepa L. seed treatment. But with soil application, the Trichoderma spp. dispersed in a greater area whereas with seed treatment the Trichoderma spp. remained concentrated on or around the seed and later on in the rhizosphere. The Trichoderma spp. receive wide range of nutrients i.e. amino acids, carbohydrates, organic acids etc. through profuse exudation during A. cepa L. seed germination and root exudation. As a result they aggregate in close vicinity of root system and multiply. This may lead to enhancement of A. cepa L. plant growth through phosphate solubilization, hormone production [40][41][42][43] and better reduction of damping off disease through pathogen suppression by the Trichoderma spp. by shielding root zone.
The field trial was continued during yala season to evaluate the efficacy of Trichoderma spp. in managing basal rot disease of A. cepa L. The effect of Trichoderma spp. on vegetative parameters of A. cepa L. was also assessed.
Results of the current field trial showed that the L. Growth, Damping off and Basal Rot Disease Incidence and Severity in Sri Lanka inoculation of the Trichoderma asperellum alone or Trichoderma asperellum and Trichoderma virens together had major influences on reduction of A. cepa L. basal rot disease incidence, severity and enhancement of growth. The reduction of disease incidence and severity of Trichoderma spp. treatments are comparable to the standard fungicide treatments. The satisfactory control of the basal rot of A. cepa L. indicates that the used Trichoderma spp. i.e. Trichoderma asperellum, Trichoderma virens suppressed the basal rot pathogen i.e. Fusarium oxysporum f. sp. cepae effectively. Additionally, Trichoderma spp. inoculation also enhanced A. cepa L. plant growth parameters. Growth promotion activities of Trichoderma might be a direct consequence of colonization, enhanced positive interaction with the plant, increased nutrient uptake by plant or due to reduction of pathogen activity [14]. Trichoderma harzianum inoculated pigeon pea (Cajanus cajan L Millsp) seedlings are reported to show increased dry weight and P uptake per plant over control [44]. Additional evidence for phosphate solubilization has been reported in studies by [25] and [14]. Induction of systemic resistance in plants by Trichoderma spp. has also been reported by many workers but these aspects were not investigated in the present study.

Conclusions
Trichoderma virens and Trichoderma asperellum isolated from soils of A. cepa L. growing areas can be effectively used in the management of A. cepa L. damping off and basal rot diseases under field condition. Further, application of Trichoderma spp. i.e. either Trichoderma asperellum alone or Trichoderma asperellum in combination with Trichoderma virens as seed coating or soil inoculation at the time of sowing and subsequent treatment with Trichoderma spp. as seedling root dip or soil infestation at the time of transplanting significantly increased growth parameters compared with that in the non-Trichoderma spp. inoculated control treatments.