Effect of Inoculum Density of Stromatinia cepivora on the Ability of Sclerotial Mycoparasites to Suppress White Rot in Garlic

White rot, an garlic disease caused by the soil-borne fungus S. cepivora, is a serious problem of garlic productions in Egypt. This study examines the potential of controlling the disease biologically by using three sclerotial mycoparasites i.e., Chaetomium globosum (Chg6), Clonostachys rosea (Cr12) and Penicillium oxalicum (Po9) employed either alone or in combinations. In in vitro assays, these sclerotial mycoparasites showed high antagonistic effect against S. cepivora isolate (Sc8). In greenhouse experiments, the chemical treatment of tebuconazole was the most effective, with the lowest incidence of white rot in garlic compared to the control. Sclerotial mycoparasites either alone or in combinations significantly reduced the incidence of white rot in garlic. In general, dual and triple combinations of the sclerotial mycoparasites were more effective than these isolates used individually. In field experiments, under low (40 sclerotia/kg of soil) and high (600 sclerotia/kg of soil) inoculum levels, the standard fungicide programme gave statistically significant white rot control, decreasing disease incidence by 67.7 & 32.4% in 2016/2017 season and 72.6 & 31.1 % in 2017/2018 season, respectively. Under low inoculum levels, significant control, equal to the fungicide treatment, was achieved with the triple combination of three sclerotial mycoparasites. However, no sclerotial mycoparasites employed alone give significant control of garlic white rot under high inoculum levels. The triple combination of three sclerotial mycoparasites decreasing disease incidence by 70.8 &25.9 % in 2016/2017 season and 73.7 & 27.6 % in 2017/2018 season, under low and high inoculum levels, respectively. The activities of defense enzymes, i.e. peroxidase, polyphenoloxidase and chitinase due to application of sclerotial mycoparasites were enhanced in garlic plants either grown under low or high inoculum levels. Reduction of white rot disease incidence was accompanied by increasing growth parameters and bulbs yield of garlic plants grown under field conditions. These results concluded that the performance of sclerotial mycoparasites may be influenced as much by the absolute disease pressure. At the low disease pressure site, the low level of S. cepivora inoculum enabled sclerotial mycoparasites to bring about disease control.


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. The annual world garlic production is about 22.2 million tons [1]. In 2016, Egypt produced approximately247,000 metric tons garlic from 10,473 hectares [2]. Unfortunately, in Egypt and in several other countries, garlic is liable to infection by several soil-borne fungi which affected on both quantity and quality of the cloves after harvest [3]. White rot disease caused by the soil-borne pathogen Stromatinia cepivora (Berk). Whetzel, is one of the most important and destructive diseases of garlic and is prevalent worldwide [4,5]. Stromatinia cepivora produces no functional spores. Instead, it propagates by the production of round, poppy-seed-sized (0.3-0.6 mm in diameter), and black sclerotia on the roots of decayed host plants. Sclerotia of S. cepivora spread via mass movement of soil or water and especially on infested plant parts. Once introduced into an area, S. cepivora is moved gradually from field to field on contaminated equipment or planting materials; slowly, the production of garlic in the entire region is threatened. Furthermore, once a field is infested, it will remain so for at least 40 years and probably longer because sclerotia of the fungus remain dormant indefinitely in the absence of Allium hosts [6]. Garlic culture is perhaps the principal mode of movement because it is propagated vegetatively, and garlic bulbs and cloves are sufficiently large that an infestation by mycelia or a few sclerotia might go unnoticed. Initial infestations of S. cepivora in afield usually are limited to patches of less than a few hundred plants when the disease is first noticed. In subsequent Allium crops, inoculum density increases, and disease incidence increases [7]. Soil temperature and seasonal time of planting also are important determinants of final disease incidence [8]. The severity of white rot is related directlyto the number of sclerotia in the soilat planting [7]. An inoculum density of a few sclerotia in a liter of field soil potentially can results in crop failure [7]. Loss estimates are difficult to ascertain because once white rot is identified in a field, growers are forced to grow other nonsusceptible (non-Allium) crops. Hence, infested fields often are abandoned from further garlic production.
No field treatment has yet been developed to completely eradicate the fungus from soil. There are few effective chemicals or other methods to control garlic white rot [9], and host resistance is not sufficient to provide commercially acceptable control [10]. As a result, attempts to manage the disease have focused on reducing the populations of sclerotia in the soil. In previous study, on onion, the fungal isolates Chaetomium globosum (Chg6) Kunze, Clonostachys rosea (Cr12) (Link) Schroers and Penicillium oxalicum (Po9)Currie & Thom were confirmed as antagonists of Stromatinia cepivora onion isolate (Sc2) and parasitized on their sclerotia [11]. We also found that in dual culture with S. cepivora, these isolates produced inhibition zones and colonized pathogen hyphae. When agar was amended with culture filtrates of Ch. Globosum (Chg6), Cl. rosea (Cr12) and P. oxalicum, the growth of S. cepivora was distorted or unusual, indicating the production of antibiotics [11]. Therefore, this paper reports the results of tow field trials which compared the standard fungicide programme for garlic white rot withsolid substrate formulations of Ch.globosum (Chg6), Cl. rosea (Cr12) and P. oxalicum employed alone or in combination applied at sowing under varying disease pressures.

Garlic White Rot Pathogen
One isolate of S. cepivora (Sc8) was obtained from the author's collection. This isolate was found to be of high virulence against garlic based on pathogenicity tests conducted in previous studies [5].

Testing the Antagonistic Activity Against S. Cepivora (Sc8)
Each of the Ch. globosum (Chg6), C. rosea (Cr12) and P. oxalicum (Po9) was tested for antagonism for S. cepivora (Sc8) using the dual culture techniques [12]. PDA plates were inoculated on one side with a 5 mm mycelial disk from a 7day-old culture of the test fungus. The opposite side was inoculated with a disc of S. cepivora (Sc8) and the plates were incubated at 18±2°C in the dark. Four replicate plates were inoculated for each test fungus. After 14 days, the following parameters were measured: (i) average growth area (cm 2 ) in both dual culture and control and (ii) average number of sclerotia (in cm 2 ) in both dual culture and control and (iii) average area of inhibition zone.

Parasitism of Sclerotia
Sclerotia of garlic pathogen S. cepivora (Sc8), produced in culture by the method of Crowe et al., [7], were surface sterilized in 1 % sodium hypochlorite for 4 min, rinsed in sterile distilled water for 5 min and placed around the periphery of 7 -day-old colonies of the test fungi grown on water agar plates. Plates were sealed to prevent desiccations and incubated at 18 ± 2°C. After 3 weeks, sclerotia were examined microscopically for evidence of parasitism. Uninfected sclerotia and sclerotia naturally infected with each of Ch.globosum (Chg6), C. rosea (Cr12) and P. oxalicum (Po9) were also examined microscopically. A sample from each group of parasitized sclerotia was removed and transferred to fresh PDA to determine their viability.

Inhibitor Effect of Cultural Filtrate Against Growth of S. cepivora (Sc8)
The effect of culture filtrates of sclerotial mycoparasites on radial growth of S. cepivora (Sc8) isolates was tested using PDA (potato dextrose agar) plates amended with culture filtrates of the tested sclerotial mycoparasites [11]. Potato dextrose broth (PDB) medium (1 L; pH 6.5-6.8) containing 200 g potato and 20 g dextrose was prepared and 100 ml were distributed into 250 ml clean flasks and autoclaved. A 5 mm mycelial disk from a 7-day-old culture of the test sclerotial mycoparasite was added to each flask. Three replicate flasks were prepared. The flasks were incubated on a rotary shaker at room temperature for 10 days. The cultures were then filtered off under vacuum and the filtrate centrifuged at 2500 g for 20 min. Twenty-five ml of the supernatant were re-filtered through a sterile 0.22 um Millipore filter directly into 225 ml molten PDA. The amended PDA was poured into Petri dishes and after cooling, the plates were centrally inoculated with a 5 mm diam. mycelial disc from the edge of a 7-day-old colony of S. cepivora (Sc8). Un-amended PDA medium, prepared as above but without the filtered culture supernatant, was used as a control. Five replicate plates with each filtrate were made. The plates were incubated at 18±2°C for 14 days. Qualitative analysis of any changes in growth form of S. cepivora (Sc8) was recorded visually: (-) no change, (+) colony compact and dense, hyphae more branched and no sclerotia formed, and (++) no growth. Tebuconazole (commercialized as Folicur ®, 25% ai. Bayer Group Science, Germany) was used in the above experiments for comparison. The chemical treatment was applied at the recommended rate of 1.0 ml Folicur/ L.

Inhibitor Effect of Cultural Filtrate Against Sclerotial Germination
Sclerotia were collected from S. cepivora (Sc8) cultures (60-day-old) and soaked in test tubes containing the culture filtrate of each of the sclerotial mycoparasites to be tested for 12 h at room temperature. At end of the soaking period, sclerotia were washed with sterile distilled water and thirty sclerotia from each treatment were transferred individually under aseptic conditions to Petri-dishes containing PDA. Water-soaked sclerotia were used as the control treatment. Four replicates (dishes) were used for each treatment. Petri dishes bearing sclerotia were incubated at 18-20°C for 7 days and percentages of germinating sclerotia were determined. Tebuconazole was used at the rate of 1.0 ml Folicur/ L for comparison.

Preparation of Sclerotial Mycoparasites Inocula
Ch. globosum (Chg6), C. rosea (Cr12) and P. oxalicum (Po9) used in this study were grown on sterilized (121°C for 60 min) wheat bran as a carrier preparation [13]. Carboxymethyl cellulose (1%) was used as an adhesive at the rate of 1:10 (v/w). The pH was adjusted to neutral by adding CaCO 3 at the rate of 15 g/kg. Mannitol was added as osmoticant at the rate of 8.5 ml of 3% mannitol for 100 g formulation. Spore suspension (10 6 spores/ml) of each of Ch. globosum (Chg6), C. rosea (Cr12) and P. oxalicum (Po9) were incorporated into sterilized wheat bran under aseptic conditions at the rate of 50 ml of suspension per 100g and thoroughly mixed with a sterilized spoon. The materials (35% moisture content) were packed in polythene bags, sealed and stored at room temperature. These formulations used alone or their dual and triple combinations (equal volumes of each) were applied in the greenhouse and field experiments.

Preparation of Garlic White Rot Pathogen Inocula
Fungal mass of garlic isolate S. cepivora (Sc8) for soil infestation in greenhouse experiments was obtained by growing the isolate on a sand-barley medium [14]. This medium was prepared by mixing 50g barley grains, 50 g sand and 40 mL water; then the mixture in glass bottles (500 mL capacity) with cotton plugs was sterilized at 121°C for 30 min. The autoclaved medium was inoculated with a 5mm disk of S. cepivora (Sc8) and incubated at 18 ± 2°C for 5 weeks.

Effects of Sclerotial Mycoparasites on Garlic White Rot Disease Development
The effects of the three sclerotial mycoparasites i.e., Ch. globosum (Chg6), C. rosea (Cr12)and P. oxalicum (Po9)and their combinations (equal volumes of each) were investigated on the development of white rot disease on garlic in soil artificially infested with S. cepivora (Sc8). The experiment was carried out in pots under greenhouse conditions (the minimum and the maximum temperatures were 5-10°C and 20-25°C, respectively) using susceptible cultivar of garlic as in El-Sheshtawi et al. [15]. The experiments were conducted with a completely randomized design (CRD) with nine treatments (seven of the three selected sclerotial mycoparasites and their combinations of Ch. globosum (Chg6), C. rosea (Cr12) and P. oxalicum (Po9), Chg6 +Cr12, Chg6 +Po9, Cr12 +Po9, Chg6+Cr12 +Po9, bioformulated on wheat bran previously described, chemical fungicide and infected control) each with four replicates. Each replicate consisted of a sterilized plastic pot (30 cm diameter) containing 5 kg of autoclaved loamy clay soil pre-infested with S. cepivora (Sc8) at the rate of 2% (w/w) 2 weeks before sowing. The formulated sclerotial mycoparasites were added (at the rate of 1% w/w) to the infested soil one week before planting. The chemical treatment was applied according to the standard fungicide programme (garlic cloves dipped in 1.0 ml Folicur plus foliar spray). The chemical treatment was applied according to the standard fungicide programme. In this programme garlic cloves were dipped and sprayed (two time intervals 6 weeks in between) with 1 ml/L Folicur. Before chemical treatment, cloves were superficially disinfected by dipping in sodium hypochlorite solution (0.25%) for 2 min and rinsed after surface-sterilization with sterile distilled water. Five surface disinfected garlic cloves (cv. Sides 40) were sown in each pot. Nitrogen fertilizer in form of urea (46% N) was added at the rate of 10 g/pot 30 days after planting and plants were irrigated when necessary. The percentages of disease incidence were calculated after 100 days after planting as follows: Disease incidence (%) = 100 × No. of infected plants/No. of total plants [16].

Selection of Trials Location
Field trials were located in El-Deer village, El-Qalubia governorate in which white rot disease was of high commercial interest. In this region, several fields with a wellestablished history of white rot disease were sampled preliminarily for inoculum levels determinations according to the procedure of Utkhede and Rahe [17]. After that, two field sites were chosen. One of them was characterized by their low sclerotial density and had an average of 40 sclerotia per 1 kilogram soil. The second was characterized by high sclerotial density and hadan average of 600 sclerotia per 1 kilogram soil.

Effects of Trichoderma Species on Garlic White Rot
Disease Development Two field trials were used to estimate the efficiency of the three selected sclerotial mycoparasites and their combinations for controlling white rot disease of garlic plants. The low sclerotial density trial had an average of 40 sclerotia per 1 kilogram soil and the high sclerotial density second trial had 600 sclerotia per 1 kilogram soil. For each trial, the experiments were conducted with a completely randomized design (CRD) with nine treatments (seven of the three selected sclerotial mycoparasites and their combinations of Ch. globosum (Chg6), C. rosea (Cr12) and P. oxalicum (Po9), Chg6 +Cr12, Chg6 +Po9, Cr12 +Po9, Chg6+Cr12 +Po9, bioformulated on wheat bran previously described, chemical fungicide and control) each with four replicates plots. The plot area was 3.0×3.5 m (10.5 m 2 ) each plot included 6 rows (each 3.0 m length and 50 cm width). Each treatment preparation freshly prepared was incorporated to soil at the rate of 300 g formulation/ m length of the row. A cavity 15-cm in depth was made in the surface of each row. Then the powder preparation of each treatment was added to this cavity and then recovered with the soil and immediately irrigated. One week after incorporation, surface disinfected garlic cloves (cv. Sides 40) were planted in each row. Garlic cloves that had been uniformly sized were hand planted 3 inches deep in rows spaced10 cm × 10 cm within each row. Based on garlic production regimes, the plots were planted with garlic cloves in 15-September of 2016 of 2016/2017 growing season and the experiment was repeated in 2017/2018 growing season. The chemical treatment was applied by dipping garlic cloves before sowing in fungicide formulation (1.0 ml Folicur/L) for 5 min. One month later, stem bases of garlic plants were sprayed (tow times) with the same concentration of Folicur at 6-weeks intervals. Irrigation and fertilization for garlic were conducted with commercial production in the area. White rot disease evaluations were conducted periodically during the growing season based on top symptoms of white rot, and were confirmed by gently removing some soil from around the base of some plants. At harvest, bulbs with symptoms of white rot were assessed by pulling and observing all garlic bulbs in each plot. The percentage of infected plants as well as white rot reduction (%) was calculated according to Hovius  White rot reduction % = White rot % in control − White rot % in treatment White rot % in control × 100

Effect of Sclerotial Mycoparasites on Enzymatic Activities in Garlic Plants
The effect of Ch. globosum (Chg6), C. rosea (Cr12) and P. oxalicum (Po9) alone and their dual and triple combinations on the activities of the defense enzymes of peroxidase, polyphenoloxidase and chitinase of garlic plants was estimated at 100 days after planting. To extract the enzyme, garlic-leave samples (g) were homogenized with 0.2 M Tris HCl buffer (pH 7.8) at 0°C containing 14 m M Bmercaptoethanol at the rate of 1/3 w/v. The extracts were obtained by filtering off the debris with a clean cloth and centrifuging at 3,000 rpm for 15 min. The supernatants were recovered and kept in a tube in an ice bath until assayed. The supernatant was used to determine the activity of enzymes using UV spectrophotometer. Peroxidase activity was assayed with guaiacol as the hydrogen donor as described by Hammerschmidt et al. [19] and peroxidase activity was expressed as the increase in absorbance at 470 nm/g fresh weight/ minute according to the method described by Lee [20]. Polyphenoloxidase enzyme activity was determined by measuring the rate of quinone formation as a result of oxidizing 3,4-dihydroxyphenylalanine (DOPA) and polyphenoloxidase activity was expressed as the increase in absorbance at 475 nm/g fresh weight/minute according to the method described by Bashan et al. [21]. The determination of chitinase enzyme was carried out using colloidal chitin as substrate and dinitrosalicylic acid (DNS) as reagent to measure reducing sugars according the method described by Monreal and Reese [22]. Chitinase activity was expressed as mM N-acetylglucose amine equivalent released/ gram fresh weight/ 60 minutes at 450nm.

Effect of Sclerotial Mycoparasites on Plant Growth and Bulb Yield
The effect of Ch. globosum (Chg6), C. rosea (Cr12) and P. oxalicum (Po9) alone and their dual and triple combinations on plant growth were studied on onion and garlic grown under field conditions. The isolates alone and their dual and triple combinations and chemical fungicide were applied as described in field experiments. Four replicates were used per treatment. Negative control plots were treated with wheat bran free of antagonistic fungi. At 100 days after planting, some vegetative growth parameters: average plant height (cm), average number of leaves/plant and average plant biomass (g), of each crop was estimated. At the end of the experiment (180 days for garlic after planting), fresh weight of onion and garlic plants (bulbs with the tops of the plants) within each plot were weighed. Efficacy of treatments was calculated using the following formula: Efficacy (%) = Fresh weight of plants in control-Fresh weight of plants in treatment/ Fresh weight of plants in control × 100.

Statistical Analysis
Data were entered into SPSS software version 14.0 and analyzed statistically by the analysis of variance test (ANOVA) and the means were compared by Duncan's multiple range test at P< 0.05. Data collected from field experiments were analyzed separately for each growing season. Data for percentage germinated sclerotia and percent data on disease incidence were statistically analyzed after arcsine square-root transformation; however, untransformed data are presented.

Parasitism of Sclerotia
The isolates of Ch. globosum (Chg6), C. rosea (Cr12) and P. oxalicum (Po9) were seen to colonize the surface of sclerotia of S. cepivora (Sc8) ( Table 1). The inner tissues of sclerotia appeared degraded and resulted in collapse of the outer rind. Parasitized sclerotia failed to germinate when they were transferred onto fresh PDA plates indicating that the sclerotia of S. cepivora (Sc8) had been killed by these mycoparasites.

Inhibitor Effect of Cultural Filtrate to S. cepivora (Sc8) Growth
Culture filtrates of sclerotial mycoparasites had significant effects on the growth of S. cepivora (Sc8) ( Table 2). P. oxalicum (Po9) was the most effective followed by Ch. globosum (Chg6) and C. rosea (Cr12). Filtrates of these isolates were fungistatic. The filtrates caused substantial reduction in growth rate and also caused changes in growth form. Culture filtrates of P. oxalicum (Po9) was the most effective followed by Ch. globosum (Chg6), inhibiting the growth of S. cepivora (Sc8) by 90.8 & 85.0% and 87.2 & 80.8%, after 7 and 14 days of inoculation respectively. The chemical treatment with tebuconazole completely inhibited the growth of S. cepivora (Sc8) after both durations. The inhibited colonies of S. cepivora (Sc8) after 14 days were usually compact and dense, the hyphae were generally more branched and no sclerotia were formed. Obtained data are in agreement with those reported by Harrison and Stewart [23] and by Elshahawy et al. [11]. Ch.globosum, C. rosea (syn. Gliocladium roseum Bainier) and P. oxalicum have been shown to be biocontrol agent against several soil-borne plant pathogens [24,25]. Tathan et al. [26] reported that Ch. globosum inhibited the spore production of Bipolaris oryzae (Breda de Haan) Shoemaker by 87.94%. Characterization of antifungal metabolites of Ch. globosum was studied by Biswas et al. [27] and they reported that five metabolites produced by Ch. globosum of which two metabolites, viz. chaetoglobosin and chaetomin proved effective in suppressing the growth of

Greenhouse Experiments
The chemical treatment was the most effective, with the least percentage of white rot disease in garlic (20.0% in 2016/2017 season and 25.0% in 2017/2018 season), in comparison with 100% for the control ( Table 4). The sclerotial mycoparasites (either individually or in combination) significantly reduced the incidence of white rot on garlic. In general, dual and triple combinations of the sclerotial mycoparasites were more effective than these isolates used individually. The combination of all three sclerotial mycoparasites was the most effective treatment, decreasing disease incidence by 60.0 in 2016/2017 season and 55.0% in 2017/2018 season. The obtained reduction in invaded garlic plants with S. cepivora (Sc8) may be attributed to the high accumulative inoculums potential of the introduced mycoparasites into the root region, before sowing and throughout the growing season as well, where they have a direct impact on sclerotia population. Similar explanation was reported by Kay and Stewart [31].

Field Experiments
Results of the two trials followed the same trends, but the amount of white rot was related to inoculum density. The mean disease incidence and severity of S. cepivora infection among plants in soil containing 40 sclerotia/kg of soil were significantly less than in those containing 600 sclerotia/kg of soil. In general, the sclerotial mycoparasites were more effective in reducing garlic white rot disease in the trial with low inoculum density than in high inoculum density (Table  5). In the low inoculum density trial, the chemical treatment was the most effective, with the lowest disease incidence  (Table 5). In general, the dual and triple combinations of the tested sclerotial mycoparasites isolates were more effective than these isolates used individually. The combination of all three sclerotial mycoparasites was the most effective treatment, decreasing disease incidence by 70.8% in 2016/2017 season and 73.7% in 2017/2018 season. Such results are in agreement with the earlier findings of Kay and Stewart [31] and Elshahawy et al. [11]. These results also agree with those reported recently by many researchers [25,32,33]. They reported that Chaetomium globosum, P. oxalicum and C. rosea (syn. Gliocladium roseum Bainier) reducing damage caused by seed rot and damping off, of several seed-and soilborne plant pathogens. In New Zealand, under controlled conditions, Ch. globosum provided an average of about 73% suppression of onion white rot over two years [31]. They confirmed that the use of Ch. globosum is as effective as Trichoderma harzianum Rifai and Trichoderma viride Pers. Penicillium oxalicum reduced vascular wilts caused by Verticillium dahliae Kleb. and Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyder & H. N. Hansen under glasshouse and field conditions [25]. Hoitink and Boehm [34] have reported several types of mechanisms that are used by biocontrol agents. These include competition for nutrients and ecological niches, parasitism and production of cell-wall hydrolytic enzymes and/or of antifungal compounds.

Effects on Enzymatic Activities in Garlic Plants
Results of the two trails followed the same trends. The tested sclerotial mycoparasites used either individually or in combination were pronounced in induction of defense enzyme in comparison with the control. In general, the dual and triple combinations were more effective than used these isolates individually ( Table 6). The triple combination of all sclerotial mycoparasites was the most effective treatment, induced high activation of peroxidase, polyphenoloxidase and chitinase by 70.3; 67.3, 59.9; 61.0 and 56.6; 60.6% increase over control, under low and high inoculum density, respectively. A positive correlation between the biocontrol activates of sclerotial mycoparasites isolates and enhancement of peroxidase, polyphenoloxidase and chitinase enzymes in garlic to resist infection with S. cepivora. The reduction in garlic white rot disease incidencemay is due to an increase in the defense-related enzymes such as peroxidase, polyphenoloxidase and chitinase. These results also are in agreement with those obtained by De Cal et al. [35]. They reported that induction of resistance in tomato plants was demonstrated as the main mode of action of P. oxalicum against Fusarium oxysporum f. sp. lycopersici. They added that this resistance was also observed both in sensitive and resistant cultivars indicating the role of general resistance mechanism. Recently, Aggarwal [36] investigated the important parameters of induced resistance in wheat (Triticum aestivum) against B. sorokiniana and Puccinia recondita Roberge ex Desm. using biocontrol agent C. globosum. Enhanced activities of defense related enzymes polyphenoloxidase, peroxidase, phenylalanine lyase and catalase revealed the role in induction of systemic resistance. The results indicate that the biocontrol agent induced effective defense responses in wheat plants against B. sorokiniana and P. triticina. The reduced disease incidence in wheat by Ch. globusum may be a result of cell wall strengthening through deposition of lignin and induction of defense enzymes. The oxidative enzymes play an important role in induced resistance by the oxidation of phenols to oxidized toxic products (quinine) which limit fungal activity. Peroxidases catalyze a number of reactions that fortify plant cell walls. These reactions include the incorporation of phenolics into cell walls and lignifications and suberization of plant cell walls. On the other hand, the chitinase enzymes play roles in plant defense against fungi by hydrolyze their cell wall. The amount of them significantly increase and play main role of defense reaction against fungal pathogen by degrading cell wall, because chitin is a major structural component of the cell walls of many pathogenic fungi [19,37,38,39]. garlic growth in the trial with low inoculum density compared with the trial with high inoculum density. In the low inoculums density trial, the triple combinations of the tested sclerotial mycoparasites isolates was the most effective, increasing average plant height by 23.9%, increasing average number of leaves/plant by 31.5%, increasing average plant biomass by 40.1%, respectively ( Table 7). The effects of soil application with sclerotial mycoparasites and their combinations on garlic bulb yield at two sites followed the same trend but the bulb yield was greater in soil with low inoculum density than with high inoculum density (Table 8). In general, the dual and triple sclerotial mycoparasites combinations were more effective than these isolates used individually (Table 8). P. oxalicum (Po9), Ch. globosum (Chg6) and C. rosea (Cr12) are well known from previous studies as plant growth-promoting agents. The ability of Penicillium oxalicum to improve the seed germination and seedling growth of cabbage has already been confirmed by Teshima and Sakamoto [40]. Fungal biocontrol agents enhanced the plant growth parameters by plant growth-promoting (PGPF) effects. Clonostachys rosea (syn. Gliocladium roseum Bainier), Penicillium oxalicum andCh. globosum influence plant growth through numerous mechanisms, which mainly include enhancing the solubilization of soil nutrients [41,36], increasing root length and number of root hairs to explore larger spaces of soil to absorb nutrients [42]and improving the production of plant stimulatory compounds, such as growth hormones, i.e. indole acetic acid, cytokinin, gibberellins, and zeatin [43][44][45][46].

Conclusion
The obtained results suggest that the potential for reducing white rot in garlic by soil application with inoculants containing sclerotial mycoparasites i.e., Chaetomium globosum (Chg6), Clonostachys rosea (Cr12) and Penicillium oxalicum (Po9) is absolutely influenced by the level of S. cepivora inocula. Under low inoculums levels (40 sclerotia/kg of soil) these sclerotial mycoparasites either alone or in combination may be useful for disease management. However, under high inoculums levels (600 sclerotia/kg of soil) triple combinations only give significant control of white rot. Reduction of white rot disease was accompanied by increasing growth parameters and bulb yield of garlic plants grown under field conditions.