Materials And Methods

This research was conducted in the Agricultural Microbiology Laboratory of Tibaitatá Research Center of the Colombian Corporation for Agricultural Research (Corporación Colombiana de Investigación Agropecuaria, AGROSAVIA).

Plant material. Healthy purple garlic bulbils obtained from a commercial crop were used. Before using the seed, it was washed with a 2% sodium hypochlorite (NaCl) solution for three min and then washed three times with sterile water.

Microorganisms. The pathogen used in the experiment was an S. cepivorum S10 isolate obtained from a garlic plant with disease sings collected from a plot of infected plants in a garlic field in Sopo municipality (Department of Cundinamarca). The pathogen’s inoculum was obtained from sclerotia produced in and harvested from a potato-dextrose-agar (PDA Oxoid^Ò) culture medium incubated at 18 °C for 24 days. Sclerotia were harvested by scraping the culture medium and stored in Falcon tubes; when used, they were washed with 2% NaCl and dried at 30 °C for 24 h. Regarding the antagonistic microorganisms, AGROSAVIA’s Microorganism Germplasm Bank provided T. asperellum Th034 and P. fluorescens Pf014 strains, which were then sown in a PDA culture medium and Luria Bertani (LB) broth, respectively. Tricotec^® biofungicide based on T. koningiopsis Th003 strain and one formulation prototype based on the B. amyloliquefaciens Bs006 strain (developed by Agrosavia) were also used. The inoculum consisted of suspensions in water at concentrations of 1x10⁶ conidia mL^-1 for fungi, and 1x10⁸ cells mL^-1 for bacteria.

Essential oils. Based on a literature review, essential cinnamon, clove, eucalyptus, lemon, oregano and thyme oils were selected because of their potential to control S. cepivorum. Essential oregano oil was produced in Agrosavia (previously known as Corpoica) by ^{Betancourt et al. (2012}), and the other oils were obtained from commercial concentrates.

Activity on sclerotia degradation. To determine the ability of antagonistic microorganisms to degrade S. cepivorum sclerotia, a modified version of the methodology described by ^{Clarkson et al. (2002}), was used, by placing 100 sclerotia in 1 cm² muslin bags, which were later placed at a depth of 2 cm in 6-ounce plastic containers filled with a sterile mixture of soil and sand (3:1). This substrate was immediately inoculated with 10 mL of the suspension of each microorganism at the previously described concentrations. During the evaluation period, the containers were irrigated every other day to keep the soil moist.

After 30 days, the muslin bags were collected; 30 sclerotia were taken from each and sown in petri dishes containing PDA medium culture or Luria Bertani (LB) agar, according to the treatment that was used. Then, the petri dishes were incubated at 18 °C for six days in order to determine their viability or parasitism. The remaining sclerotia were observed under a stereoscope to identify any damage in their structure. The trial was conducted in a greenhouse at 23 °C using a completely randomized design where the experiment unit consisted of one container with one bag with three replications; sclerotia with no treatment were used as a control.

Effect of essential oils on S. cepivorum in vitro growth. Essential oils were evaluated at three concentrations (Table 1) with Tebuconazole (1 L /200 L) as a chemical control and one absolute control. For this, the PDA was supplemented with each oil at the specified concentration, placed in petri dishes, and then sown in a 5-mm agar disk with S. cepivorum mycelium (obtained from a 12-day PDA culture medium) in the middle of the dish, incubated at 18 °C for 12 days, a period during which the percent inhibition of the mycelial growth (ICM) of the colonies in the presence of oils was recorded. The percentage was calculated in relation to the control once the fungus completely covered the petri dish. The calculation was made using the formula of Philippe et al. (2012) cited by ^{Gakuubi et al. (2017}), ICM (%)= dc - dt / dc x 100; where: dc= diameter of the pathogen’s colony in the control, and dt= diameter of the pathogen’s colony in the treatment.

Control of S. cepivorum under controlled conditions. The evaluation was performed in 0.5-L plastic pots in which one garlic bulbil was sown. The concentration of S. cepivorum used was 200 sclerotia per gram of soil (selected from previous unpublished studies because the level of mortality obtained was 83%). The bulbils were immersed in the suspensions of microorganisms, essential oils and Tebuconazole for 10 min, and then sown in pots to which 20 mL of each suspension were added in drench, according to the treatment. Two more applications in drench were applied every 15 days and the experiment was continued for 90 days (although the crop cycle lasts between five and six months, the disease may occur at any time). The experiment was established using a completely randomized design and the experiment unit consisted of eight pots with three replications. A control inoculated with the pathogen and one non-inoculated control were kept in a mesh house and irrigated daily at an average temperature of 20 °C. The evaluated variables were disease incidence (number of plants with symptoms over the total number of plants multiplied x 100) and the disease mortality (number of dead plants over the total number of plants multiplied x 100).

Statistical analysis. The data of each bioassay were subjected to an analysis of variance (ANOVA) and the means were compared using Tukey’s test (p£0.05) and the Statistix 10.0 statistical software.

Results

Activity on sclerotia degradation. Using the sclerotia that were recovered and sown in culture medium, colonies of the antagonists were obtained. In the Trichoderma strains, growth was observed from 100% of the sclerotia, while in the bacteria, growth was observed from 95% of the sclerotia. No S. cepivorum growth was observed in any of them (Figure 1A). Regarding the sclerotia of the control, they did not show infection when germinated in PDA, and S. cepivorum colonies were obtained. On the other hand, observation of the sclerotia of each treatment under a stereoscope showed that all the antagonists caused partial degradation of sclerotia, because of the loss of their structural stability (Figure 1B) which prevented the pathogen from growing from those structures.

Figure 1. A. Antagonist growth from inoculated sclerotia. B. Sclerotia degradation caused by the antagonists. Control (treatment with no application of antagonists), Tricotec (biofungicide based on T. koningiopsis Th003 strain), Th034 (T. asperellum Th034 strain), Bs006 (B. amyloliquefaciens Bs006 strain), Pf014 (P. fluorescens Pf014 strain). 

Effect of essential oils on S. cepivorum in vitro growth. In PDA agar supplemented with essential clove and lemon at the three concentrations, and thyme oils (200 and 400 ppm), growth inhibition was 2-12%, with no significant differences compared to the control. Conversely, in PDA agar supplemented with essential eucalyptus and oregano oils (200 and 250 ppm) and Tebuconazole, growth inhibition was higher than 92%; with essential cinnamon, oregano (150 ppm) and thyme oils (800 ppm), growth inhibition was 50-86% (Figure 2).

Figure 2. Inhibition of S. cepivorum growth in a culture medium supplemented with essential oils after 12 days of incubation at 18 °C. Values with the same letters do not have significant differences according to Tukey’s test (*=P>0.05). 

Control of S. cepivorum under controlled conditions. The evaluated treatments showed they could control the pathogen, except for essential oils, particularly eucalyptus oil, which had a negative effect on bulbils (data not shown), apparently due to a phytotoxic effect that caused necrosis on the tissue and affected germination. When oregano oil was applied, the performance was similar to the pathogen’s treatment, and even caused significant statistical differences (Figure 3).

Figure 3. Incidence, mortality and healthy garlic plants per treatment after being evaluated for 90 days. Columns with the same letter are not significantly different according to Tukey’s test (*= P>0.05). 

The lowest level of disease incidence (21%) was obtained with T. asperellum Th034, as well as the greatest amount of healthy or asymptomatic plants (79%) (Figure 3), while when bacteria were applied, the performance was similar to that of the tebuconazole application, with no significant differences between them, though in numerical terms the disease incidence was lower and the percentage of healthy plants increased with the bacteria (Figure 3). On the other hand, the treatment with T. koningiopsis Th003 (Tricotec^®) was the least effective for reducing the disease incidence and mortality (Figure 3). The plants of the control that were not inoculated did not show signs or symptoms of white rot.

Discussion

Sclerotia degradation is a characteristic considered when selecting an antagonistic microorganism, since sclerotia can survive in the soil for decades, thereby limiting Alliaceae production and crop yields (^{Metcalf et al., 2004}; ^{Velásquez et al., 2012}; ^{Pérez et al., 2015}). This action is related mainly to the antagonist’s ability to produce toxic secondary metabolites and extracellular enzymes, which will make it possible for it to access to sclerotia as a source of nutrients. In species of Trichoderma genus, this action is related to microparasitism, involving production and secretion of enzymes, including β 1-3 glucanase, β 1-6 glucanase, N-acetylhexosaminidase, polysaccharides, proteases and lipases, which are involved in cell wall degradation and their production has been reported in T. asperellum and T. koningiopsis strains (^{Vinale et al., 2008}; ^{Schuster and Schmoll, 2010}; ^{Mukherjee et al., 2012}; Pérez et al., 2015; ^{Fuga et al., 2016}).

Similarly, B. amyloliquefaciens and P. fluorescens are known to produce β glucanases and chitinases, and B. amyloliquefaciens produces lipopeptides, including iturines, fengicines or fatty acids and phenols, for example, chloroxylenol, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid and pyrrole, with antifungal action against pathogens such as Sclerotinia sclerotiorum or F. oxysporum. The species P. fluorescens is known to produce hydrocyanic acid, pyrrolnitrin and siderophores responsible for antifungal activity on Sclerotium rolfsii, among others (^{Ganeshan and Arthikala, 2005}; ^{Shafi et al., 2017}; ^{Vinodkumar et al., 2017}; ^{Ley et al., 2018}).

In this regard, white rot control in this study could be related to the parasitism of sclerotia in the soil eventually mediated by the production of secondary metabolites. Particularly, it has been demonstrated that T. koningiopsis Th003 and T. asperellum Th034 have control activity on pathogens that produce sclerotia. For example, it was observed that a pre-planting treatment of potato tubers infected with Rhizoctonia solani sclerotia with two formulations based on these fungi reduced the incidence of the pathogen by 80 % compared to non-treated tubers, whose incidence was 100 % (^{Beltrán et al., 2010}).

Similarly, in the control of S. sclerotiorum and Sclerotinia minor using Tricotec^®, the incidence of white mold was reduced by 34% in commercial lettuce crops (^{Moreno et al., 2010}). For T. asperellum Th034, ^{Smith et al. (2013}) determined its ability to degrade S. sclerotiorum sclerotia. On the other hand, it has been demonstrated that B. amyloliquefaciens Bs006 and P. fluorescens Pf014 have control activity on F. oxysporum in cape gooseberry plants and increase their survival in the field, making it possible to harvest their fruits (^{Díaz et al., 2012}).

Regarding essential oils, it is necessary to bear in mind that their effect can be different on mycelium and sclerotia due to the resistance of these structures. Different studies have demonstrated the inhibitory effect that the essential oils have on phytopathogens. For example, ^{Ranasinghe et al. (2002}) described Colletotrichum musae, Lasiodiplodia theobromae and Fusarium proliferatum inhibition using clove oil at concentrations of 300-1100 ppm. Likewise, ^{Barrera et al. (2009}) reported 100 % inhibition of F. oxysporum f. sp. gladioli using clove, thyme and cinnamon oils at concentrations of 100, 150, 200, 250 and 300 ppm.

Although S. cepivorum growth was inhibited under in vitro conditions, no control of the disease was observed in the bioassay conducted in soil, particularly when oregano oil was used. This may be due to the fact that the oil did not have an effect on the sclerotia and that, when germination occurred, the oil had possibly already degraded in the soil. In contrast, the phytotoxic effect observed with essential eucalyptus oil, at the concentration applied, makes it non viable for garlic crops, although it is well know that this oil has wide-spectrum pesticide properties, since different studies have demonstrated its activity as an antimicrobial compound that affects both bacteria and fungi, and as a pesticide, acaricide and herbicide (^{Batish et al., 2008}; ^{Haouel et al., 2015}; ^{Tomazoni et al., 2017}). In this regard, it is necessary to evaluate other concentrations or application methods before using it as an alternative for controlling white rot.

Although the results of this study show that the antagonists reduce the negative impact of the disease, before using them in the field, further studies must be conducted to determine application methods and frequency, particularly during soil preparation, in order to reduce the pathogen’s inoculum, according to the results obtained by ^{Velásquez et al. (2012}), who demonstrated that the sclerotia can move from deep areas (20 to 40 cm) to the upper layer, and vice versa, during soil preparation.

Conclusions

Although essential oregano (200 and 250 ppm) and eucalyptus oils inhibited S. cepivorum growth in in vitro experiments, when applied to bulbils and the soil, oregano oil had a similar performance to the control pathogen, while eucalyptus oil had a toxic effect that affected bulbil germination. Conversely, the antagonistic microorganisms reduced the disease incidence and mortality, and T. asperellum Th034 being the most effective.