Integrated Management of Garlic White Rot (Sclerotium cepivorum Berk) Using Some Fungicides and Antifungal Trichoderma Species

Garlic (Allium sativum L.) is a monocotyledonous plant and belongs to the family Alliaceae. It is the second most widely cultivated vegetable next to onion and widely produced for its medicinal and nutritional properties and has been recognized in almost all the cultures for its culinary properties. Garlic is an excellent source of several minerals and vitamins that are essential for health and has medicinal role for centuries such as antibacterial, antifungal, antiviral, antitumor and antiseptic properties [1]. In Ethiopia, the total area under garlic production in 2011/12 reached 13,278.55 ha and the production is estimated to be over 123,961.46 tons annually [2]. Production of garlic is done on sandy soil with higher organic matter content, pH 6-7 at altitude of 1800-2500 m.a.s.l, rainfall 600-700 mm and temperature of 15-24°C [3,4]. Economic significance of garlic in Ethiopia is fairly considerable and contributes to the national economy as export commodity [5] and important for small holder farmers [6]. It was reported that heavy damage to garlic due to fungal diseases, in later years, has become very important in major production areas of garlic [7-11].


Introduction
Garlic (Allium sativum L.) is a monocotyledonous plant and belongs to the family Alliaceae. It is the second most widely cultivated vegetable next to onion and widely produced for its medicinal and nutritional properties and has been recognized in almost all the cultures for its culinary properties. Garlic is an excellent source of several minerals and vitamins that are essential for health and has medicinal role for centuries such as antibacterial, antifungal, antiviral, antitumor and antiseptic properties [1]. In Ethiopia, the total area under garlic production in 2011/12 reached 13,278.55 ha and the production is estimated to be over 123,961.46 tons annually [2]. Production of garlic is done on sandy soil with higher organic matter content, pH 6-7 at altitude of 1800-2500 m.a.s.l, rainfall 600-700 mm and temperature of 15-24°C [3,4]. Economic significance of garlic in Ethiopia is fairly considerable and contributes to the national economy as export commodity [5] and important for small holder farmers [6]. It was reported that heavy damage to garlic due to fungal diseases, in later years, has become very important in major production areas of garlic [7][8][9][10][11].
Of the fungal diseases, white rot (Sclerotium cepivorum Berk) is the most destructive disease of garlic, and other Allium species throughout the world. It attacks leaves, roots, and bulbs of Allium spp. and can survive in the soil for nearly 20 years. Sclerotia are the only reproductive structures of S. cepivorum has no perfect stage has not yet been described and no asexual spores are produced. The sclerotia are stimulated to germinate only by Allium-specific root exudates (alkylcysteine sulphoxides) which are broken down by soil microorganisms to form thiols and sulphide compounds and then stimulate S. cepivorum sclerotia to germinate, indicating that the host range is limited to Allium species [10]. White rot causes important economic losses in garlic production worldwide and can cause losses from 1 to 100% [11]. In Ethiopia, the yield loss has been found to range between 20.7% and 53.4 % [12]. Once it is established permanently renders a field unusable for a garlic production. In spite of its importance garlic productivity in many parts of the world, is low due to the lack of improved variety and, traditional production system besides diseases and pest problems. The use of low quality seeds, imbalanced fertilizers, inappropriate agronomic practices, uneven irrigations and marketing facilities are the main constraints [9,10]. Management of diseases caused by soil borne pathogens like S. cepivorum, is very difficult and need a multi-pronged management strategy [13]. The earliest methods used to control garlic and onion white rot were cultural and physical practices of field hygiene and sanitation and crop rotation were used for primary inoculums reduction. These have been viewed as impractical for Allium white rot control due to long persistence nature of the sclerotia for more than 20 years. Soil flooding, soil solarisation and sterilization, biological control agents, of all possible combinations with the objective to achieve integrated management of garlic white rot using four Trichoderma spp of PPRC isolates and two recommended fungicides [Apron Star 42 WS and Tebuconazole (Folicur 250 EC] under greenhouse condition. The Sclerotium cepivorum sclerotia propagules were maintained and undertaken in pot experiment (Seedling bioassay), as described earlier by [23,29]. Inoculated local garlic clove with S. cepivorum and un-inoculated alone were used as positive and absolute control, respectively.

Culturing of Sclerotium cepivorum
Culture specimens of S. cepivorum preserved in the Mycology Section of Ambo Plant Protection Research Centre (APPRC) were used for this study. Stock culture was inoculated onto sterile potato dextrose agar (PDA) plates and incubated at 25°C for 2 days and then examined for the growth of the fungus. After incubation, the appearance of colonies on the medium was observed which proved the viability of preserved isolates of the S. cepivorum. The well-grown mycelium was selected for further study.

Mass production of Sclerotium cepivorum
The sclerotia of S. cepivorum isolate were first produced on PDA in 9-cm diameter petri dishes by incubating at 20°C for 5 days. Since the pathogen doesn't have functional spores, a small, round, seed-like structure known as sclerotia was initially produced. The refreshed S. cepivorum sclerotia were further inoculated on whole wheat grains [30]. Fifty grams of the inoculated whole wheat grains were added to each of twenty, 250 ml conical flasks, the content of the flasks were treated with 45 millilitres of 0.0025 % (w/v) Chloramphenicol and the flasks were left overnight at room temperature. The treated flasks of wheat (50 g each) were autoclaved at 121°C and 15 psi for 30 min, and this was repeated for three consecutive days. After cooling to room temperature, each flask was inoculated with four, 5 mm disks of S. cepivorum taken from the actively grown edge of a 5 day old culture grown on PDA. The flasks were incubated at 20°C in the dark for 6 to 8 weeks and shaken at weekly intervals to ensure an even distribution of mycelium. During the first three weeks of incubation, 0.5 ml of sterile distilled water (SDW) was added if the flasks appeared dry, to encourage mycelia growth .

Harvesting Sclerotium cepivorum sclerotia
The sclerotia of S.cepivorum were harvested from the wheat grains using progressive wet sieving through 850 µm, 500 µm and 250 µm sieves [31,32]. Only healthy sclerotia was retained on the 500 µm sieve which was air dried on sterilized Whatman No. 1 filter paper for 24 h before they were used or conditioned. The sclerotia used after this stage was termed "fresh". Before using for the greenhouse study, both the fresh and conditioned sclerotia viability were resolved by taking a sample of 100 sclerotia and surface sterilized in 0.25% sodium hypochlorite (NaOCl) for 1min. Subsequently, it was washed in five changes of sterile distilled water (SDW), then spreaded over Whatman No. 1 filter paper to absorb excess liquid. Then, it was placed onto PDA in petri-dishes. The petri-dishes were sealed with polythene wrap and then incubated at 20°C in the dark and the sclerotial viability/ germination was examined for 10 days. The number of germinated sclerotia was recorded to reach >96%. Once the viability of the sclerotia germination percentage and competence were decided, 100 g of sclerotia/kg of sterilized moist soil was incorporated into the in vivo experiment. This is based on the fact that 0.01-0.1 g sclerotia/g of soil resulted in infection of less than or equal to 85-100% and 100% incidence of disease in onion and garlic plants, respectively. This is sclerotia germination stimulants (diallyl disulfides, DADS), composted onion waste, host resistant were also found moderately effective at varying degrees [14][15][16]. It has been found that systemic as well as non-systemic fungicides significantly reduced garlic white rot disease development and resulted in improved garlic yield. Several effective fungicides have been recommended against this pathogen. Among these, Tebuconazole was also effective in reducing the incidence and in increasing the yield when applied as a clove treatment [17,18].
Recent efforts have focused on developing economically safe, long lasting and effective bio-control methods for the management of plant diseases. Use of biocontrol agents has been shown to be eco-friendly and effective against many plant pathogens. Among the fungal antagonists, Trichoderma is considered as the most important because it controls various soil borne and seed diseases caused by a wide range of fungal pathogen [19,20]. Trichoderma grows rapidly when inoculated in the soil as it is naturally resistant to many toxic compounds including herbicides, fungicides and insecticides such as DDT and phenolic compounds. The resistance to toxic compounds may be due to the presence of ABC transport systems in Trichoderma strain. The biocontrol mechanisms exercised by Trichoderma could be attributed to mycoparasitism, competition for nutrients, release of toxic metabolites and extra cellular hydrolytic enzymes [21].
In Ethiopia, research effort on host resistant against garlic white rot is very limited. It was reported that systemic as well as non-systemic fungicides significantly reduced incidence of white rot, its progress rate and severity that also resulted in improved garlic yield [7,9]. Study revealed that some of the Trichoderma species are endowed with great potential in controlling the garlic white rot [22].
The most effective control systems to date have involved the integration of a number of systems for managing garlic white rot [13,23]. The combined use of biocontrol agents and chemical pesticides has attracted much attention as a way to obtain synergistic or additive effects in the control of soil-borne pathogens. Seed treatment with Trichoderma along with compatible fungicide is common practice among the farmers for economic and effective management of seed and soil-borne plant diseases. Combination of Trichoderma with reduced levels of fungicide promotes the degree of disease suppression without risk on non-target organisms similar to that achieved with full dose of fungicide application [24][25][26]. Trichoderma harzianum C52 was found to be compatible with some fungicides and determined to be effective biocontrol agent of the onion white rot pathogen [27]. It was found that T. viride combined with either Tebuconazole or onion compost resulted in enhanced white rot control (>90%) and was better than any treatment alone [23,28].
However, attempt has not been made in Ethiopia to determine the effect of integrating various control measures with Trichoderma species for the management of white rot in garlic. Hence, the present study was undertaken on the management of garlic white rot with the integration of four selected Trichoderma spp. (T. hamatum, T. harzianum, T. oblongisporum and T. viride.) and two recommended fungicides (Apron Star 42 WS and Tebuconazole) under pot culture condition. In this paper the results of this integrated management of garlic white rot under pot culture condition is described.

Experimental design
The experiment was conducted in a Completely Randomized Design (CRD) with three replications and 31 treatments consisting similar to the finding that only one sclerotia per kilogram of soil can provoke a 50%, and 10-20 sclerotia per kilogram can result in infection of essentially all plants (as the disease severity depends on sclerotia levels in the soil at the time of planting [33].

Mass production of Trichoderma spp.
The Trichoderma spp. used in this study were obtained from the culture specimen collections of APPRC, that, previously isolated from soils characterized in Ethiopia and preserved in culture collection [34]. These Trichoderma spp. were found to be effective in controlling faba bean fungal disease, Fusarium solani [35]. Furthermore, out of seven Trichoderma species tested under in vitro and in vivo antifungal activities against white rot of garlic, four of them registered high percentage inhibition zone ranging from 51.7 to 59.3%. [26] Therefore these four potent species were selected for the present study viz., T. hamantum, T. harzianum, T. oblongisporum and T. viride.
Mass multiplications of Trichoderma spp. were carried out according to standard procedures [36,37]. Thus, spore suspensions of Trichoderma spp. were prepared by adding 20 ml sterile distilled water to a three-week-old petri-dish cultures and scraping gently with a sterile spatula. The harvested spore suspension of Trichoderma spp. were inoculated into a sterilized one litre jar containing wheat bran, sand and water medium or sorghum grain and incubated for three days at 20°C.

In vivo efficacy test
The experiment was conducted under greenhouse condition using the local cultivar of garlic. The appropriate soil composition were made proportionally with the composition of sand, compost and sandy clay loam soil mixed at (1:1:2 ratios) and then sterilized. Each pot (21 cm top diameter and 9 cm height) were filled with 3 kg of mixed soil. The pots were arranged and placed in saucers so that all watering were from below, then after, the cloves of garlic were first surface sterilized using 70% ethanol for five mins and rinsed three times with SDW. Then cloves were dressed with recommended fungicides (Apron Star 42 WS (3gm of Apron Star 42 WS powder with 10 ml of water) and Tebuconazole (2.1 ml of Tebuconazole with 15 ml water) [4] by partial and/or with combinations of both fungicides and then soaked for one hour.
The treated cloves were planted at 3 cm depth into the moist soil thoroughly incorporated with 100 g sclerotia propagules/kg of soil in the pot (5 cloves/pot were planted and two of them were thinned after germination) immediately under greenhouse condition at 12-15°C minimum and 26-30°C maximum temperature. Each Trichoderma spp. spore suspension were prepared by diluting with SDW at the rate of 10 g Trichoderma spp. mass produced/2 litre of water were mixed. Subsequently, 300 ml adjusted spore suspension of Trichoderma spp were drenched on the planted soil of each pot after seven days and continued within three days intervals [38]. The emerging garlic plants were assessed for symptoms of white rot every week up to 18 weeks. The treatments were arranged as (i) four Trichoderma spp. each alone or (ii) two fungicides each alone or (iii) fungicides combined with one or more Trichoderma spp. These were evaluated for their potential to control garlic white rot on garlic under greenhouse condition. Thus, the effect of partial and combined treatments for the control of Sclerotium cepivorum was examined and the result was compared with un-inoculated treatment. White rot disease incidence and severity was recorded in each pot.
The following treatments were applied for the experiment:

Data analysis
Data on initial and final plant stand count at emergence, disease incidence and severity were collected every week from the experiment. All garlic bulbs were hand-harvested from each pot. Average of plant height, shoot length, root length and bulb biomass were recorded at soggy/moist phase and also after drying the samples in air for 7 days. Furthermore, 5 bulbs were randomly collected from which bulb diameter were measured, weight of cloves per bulb/plant were determined and number of cloves per bulb/plant were counted as described by [39]. Severity was assessed using a scale from 0 to 5 [40] and Disease Severity Index (DSI) was calculated.
Plants were uprooted separately from pots of each replication and determined for mycelium expansion; bulb and root rots were undertaken. A disease severity index based on symptoms observed and a disease severity formula was used to rate garlic treatments for their resistance to S. cepivorum. The Analysis Of Variance (ANOVA) of the data was separately subjected to SAS version 9.0 for further analysis and also the treatment mean were further separated by Duncan's Multiple Range Test (DMRT) at 5% significance level.

Effect of treatments on the foliar, stem base and bulb rot symptoms
The treated garlic plants showed slightly yellowing and wilting of delicate leaves and thin stems appeared after germination and also very few elongated roots developed on bulbs. White rot incidence was evaluated every week from the first appearance of the disease. Infected plants were examined as a small patch of plants or single plant more chlorotic than surrounding plants 45 days after artificial inoculation. The symptoms appeared as chlorosis as of lower leaves beginning at the tips, followed by a necrosis and collapse of the affected leaves of the aerial parts of the seedlings (Figure 1).
Observation of bulbs infections were carried out after harvest 127 days after artificial inoculation. Thereafter, the development of mycelia mat around stem base and sclerotial emerged on the bulbs of different treatments were seen ( Figure 2). The seedlings exhibited characteristic garlic white rot symptoms including the blueing foliage, leaf tip dieback and a patchy distribution of diseased seedlings within each pot as shown in (Figure 3).
The disease symptom was initiated early in the trial just three weeks after planting. Some seedlings were discoloured, collapsed and lying on the soil surface of the pot. White rot symptoms appeared at about 45 days after planting the inoculated garlic bulbs and it's foliar and stem base symptoms incidence assessment was recorded weekly. Initially, disease symptoms were observed in all treatments, while the disease increased slowly in treatments and then become conspicuous just at the three to five leaves stage. The observations of assessments made at three stages (i). Foliar symptoms (every week until 98 days), (ii) Stem base symptoms (84-98 days), and (iii) Bulb rots symptoms (126 days) are presented in Table 1. When there is no stunting, no leaves colour change, no chlorosis, no wilting and collapsing and no stem base rotting, the bulb is designated as "Healthy".
The diseased tissues were diagnosed and the pathogen was reisolated as an evidence of the disease development in the trial. Over 50% of the total white rot infections were recorded in the first seven weeks. The number of diseased seedlings in all treatments increased slowly for the duration of the trial and after 12 weeks. Among 279 seedlings, 126 were infected with garlic white rot. These seedlings showed characteristic garlic white rot symptoms including the blueing foliage, leaf tip dieback and a patchy distribution of diseased seedlings within each pot. After 16 weeks, more than 80% of the seedlings were diseased in the pathogen control; a significantly greater (p>0.05) amount of disease than on both fungicides treatment applications ( Table 2). In the positive control (T2), the whole plants were extremely affected within less than 6 weeks and the seedlings were completely died.

Effect of treatments on the disease incidence and severity on garlic plants
Significant differences on disease incidence was observed at all assessment times among the treatments (p>0.05) ( Table 2). The highest incidence and severity was recorded on negative and positive control (T1 and T2) (94.4% and 100%, respectively) and with both fungicides (T3, T4) (88.9%) and Apron Star 42 WS combined with T. harzianum and T. viride (T18) (77.8). Tebuconazole combined with T. oblongisporum and T. viride (T30) has (77.8%) disease incidence under similar conditions. The highest disease incidence and severity observed in uninoculated (-ve absolute control) treatment (T1) was assumed to occure from mycelia remained in the cloves after surface sterilization. Apron Star 42 WS treated with both T. hamatum and T. viride (T16) has provided efficient and highly significant disease control as compared with uninoculated check. Whereas, Tebuconazole combined with T. hamatum (T21) and T. viride alone (T8) were the next treatments that showed lower percentage (11.1%) of disease incidence as compared to all other treatments except "Apron Star 42 Ws combined with T. hamatum and T. viride". Therefore, these two treatments are relatively the promising bioagent for antagonising garlic white rot next to Apron Star 42 WS combined with T. hamatum and T.viride (T16).
It was observed that cloves/seedlings treated with Tebuconazole showed a suppressive effect on the developments of the whole plant and roots formation and even delayed the germination by a week. In all treatments receiving Tebuconazole, the plant stands was weak, very thin and fragile. The roots were very few in numbers and very thin, shrivelled, elongated and sheath paled off easily and bulbs were tiny.
The beneficial effect of the fungicide in combination with Trichoderma spp. at planting was effective to suppress the sclerotial germination. These results indicate that, combining of fungicide with the bio-agent(s) provides a good prospect for garlic growers as Trichoderma spp. are safe for animals, human beings and environment. The results indicate a break from susceptible cropping, and integration of fungicide with biocontrol agents was the strategy providing greater reduction in suppressing the viability of S. cepivorum inocula in the soil resulting in minimum incidence of white rot (T16) (Figure 6). The magnitude of economic benefit and synergistic value of Trichoderma spp. after combination remains to be determined, as does the ecological one, when combining these practices. It can be seen that, the degree of disease control achieved by treatment with Tebuconazole alone was lower than that of all the treatments combined with Apron Star 42 WS fungicide, and mixed-up of one or more mixture of four Trichoderma spp. each other when compared to un-inoculated and T16 which has absolutely controlled the pathogen.
It was reported that treatment of garlic cloves with Tebuconazole and base spray provided significant reduction in the rate of disease progress and the final of plant mortality by S. cepivorum [17]. Eighty five percent disease incidence reduction was reported in Tebuconazole treated plots compared with untreated plots in onion [46]. Other researchers also reported that combinations of Tebuconazole and a biocontrol agent enhanced the control of onion white rot [23]. Even though an indication of antagonism has been obtained from this greenhouse study and previous literature based on similar isolates [32], the level of control did vary with the same isolates when similar methodology was used. In general, an important factor in biocontrol agent effectiveness is the rate at which the propagules/mycelium dilution amounts proliferate when applied to the potting mix. To predict and successfully use biological control agents for soil borne disease control, it is critical that their biology and ecology be more completely understood. It was beyond the scope this study to determine the individual components of the three types of potting mixed-up in relation to microbial carrying capacity as an indicative difference to antagonize the white rot pathogen activities. Thus, integration of fungicides and biological control agents may enable the number of fungicide sprays to be reduced, while providing control of garlic white rot.

Conclusion
The results obtained in this study revealed that the two Trichoderma spp. (T. hamatum and T. viride) in combinations with two fungicides can have substantial antagonistic activity against garlic white rot pathogen. This could be attributed to their synergistic and additive growth effects that yielded better biomass besides controlling the disease. The findings also suggest that T. hamatum and T. viride are playing an important role in controlling garlic white rot pathogen better than the two fungicides alone. This is highly advantageous in light of the fact that the use of Trichoderma-based products is not only safe for the farmers and consumers but is also environmentally friendly. Tebuconazole has been frequently reported as effective fungicide against this aggressive pathogen worldwide, including in Ethiopia, whereas Apron Star 42 WS is reported here in Ethiopia for the second time, while it is not common elsewhere. Since the compatibility of Trichoderma spp with these two fungicides is now proved in this study, the method can be tested for control of other diseases.