MATERIALS AND METHODS

Sclerotinia sclerotiorum was obtained from the microbial collection of the Soil Microbiology Department, Colegio de Postgraduados. The fungus utilized in the present experiment was previously isolated from vegetative material of lettuce plantations at the Bajío region in Guanajuato, Mexico. In order to examine the effects of different concentrations of potassium bicarbonate (KHCO₃) on the microscopic growth of S. sclerotiorum, the Riddell’s microculture technique was used (^{Riddell, 1950}). This consisted in placing a V-shaped glass rod into glass Petri dishes which included microscopic slides and cover slips. Later, the Petri dishes were sterilized at 150 °C for 5 h. After sterilization, a 10 mm diameter-disk of Potato Dextrose Agar medium (PDA® Merck, Darmstat, Germany) enriched with each concentration of KHCO₃ (Fermont®, Monterrey, Mexico; 0, 2, 4, 6, 8, 10, 25 and 50 mM), were placed on the microscopic slides. Then, S. sclerotiorum was inoculated with a sterile needle on one side of the PDA-disk, and later, the cover slip was placed on it. This procedure was repeated twice for each KHCO₃ concentration, and a control without bicarbonate was also included.

In order to maintain the humidity in the Petri dishes, 10 mL of 10% glycerol were added. All Petri dishes were kept at room temperature conditions (~20 °C) and an approximate photoperiod of 12 h. Every day, the fungal growth was monitored under optical microscope (Leica CME, U.S.A.). Once the PDA-disk was fully covered with the fungal mycelium (approximately seven days of incubation), glycerol was replaced with a 10% formaldehyde solution, which was kept for 2 h for permanently fixing the fungal structures.

Later, the microscopic slide was removed from the Petri dish to prepare the fungal slides. The cover slip was carefully separated from the agar and placed on another clean slide on which a drop of the colorant cotton blue in lactophenol was added. The next step was to remove the PDA-disk from the original microscopic slide on which the colorant was also added, and a clean cover slip was immediately placed on it. In this way, four fungal preparations were obtained from each concentration of KHCO₃, including the control without bicarbonate. Once the excess of colorant was removed, the stained fungal preparations were sealed with colorless nail polish, and evaluated under light microscope. The microscopic evaluations consisted on identifying the growth of the sclerotial primordia in each concentration of KHCO₃. For this, an optical microscope (OLYMPUS BX51, Japan) was utilized for taking microphotographs of the fungal structures under phase contrast microscopy. Chi-square “goodness of fit” tests were performed in order to compare the effect of different concentrations of sodium bicarbonate on sclerotia formation for each one of the four structure phases. For it was used the VassarStats: Web Site for Statistical Computation (^{Lowry 2001-2018}).

RESULTS AND DISCUSSION

The presented fungal structures are part of the process of sclerotia formation by S. sclerotiorum, and were microscopically observed before the initiation stage. The sclerotial formation in the control treatment (Figure 1, A-C) began with the proliferation of primary branching of main hyphae, thus, denoting the formation of the sclerotial primordia. As second structure, the hyphal branching became more profuse, and the presence of septa was observed in apical zones (Figure 1, D-F), while the third structure was characterized by presenting small hyphal clusters (Fig. 1, G-I). Finally, in the fourth structure, a massive cluster of hyphae was observed, in which some pigmented cells were visible (Figure 1, J-L). The initiation stage began with the union of several hyphal clusters, and this stage was macroscopically observed when a hyphal conglomerate starts growing on the surface of the culture medium.

Figure 1 Microscopic developmental structures observed during the sclerotial formation of Sclerotinia sclerotiorum after seven days of fungal growth, without KHCO₃ (Control). First structure: branching of hyphae, indicated by arrows (A-C); second structure: profuse branching of hyphae (D-F); third structure: clustering of hyphae (G-I); fourth structure: massive clustering of hyphae that build up the sclerotia in the initiation process (J-L). Microphotographs were taken in phase contrast microscopy at 40X magnification. Bars = 10 µm. 

Results showed that the application of KHCO₃ had inhibitory effects on the morphology of the sclerotial primordia (Table 1). In fact results from the 50 mM dose were not included on statistical analysis due the complete absence of mycelial growth. Unlikely the treatment without bicarbonate (control) and 2 mM bicarbonate, treatments with all the others concentrations of this chemical compound showed only three developmental structures, in which we noticed that the primordia were irregularly shaped with the formation of loose cells. As the concentration of KHCO₃ increased, primordia became smaller and less compact. The increase in the concentration of bicarbonate resulted in growth inhibition of both hyphae and sclerotia (Figure 2 and 3). However, at 2 mM, 4 mM and 6 mM concentrations of KHCO₃ only scarce morphological changes were observed.

Figure 2 Microscopic developmental structures observed during the sclerotial formation of Sclerotinia sclerotiorum, after seven days of fungal growth. First (indicated by arrows), second, and third structure of sclerotial development in presence of KHCO₃: (A-C) 2 mM, (D-F) 4 mM, and (G-I) 6 mM. Microphotographs were taken in phase contrast microscopy at 40X magnification. Bars = 10 µm. 

Figure 3 Microscopic developmental structures observed during the sclerotial formation of Sclerotinia sclerotiorum, after seven days of fungal growth. First (indicated by arrows), second and third structures of sclerotial development in presence of KHCO₃: (A-C) 8 mM, and (D-F) 10 mM. For 25 mM a cellular disorganization of the primordium was observed (G-I), in which the second and third structures of sclerotial development were not observed. Microphotographs were taken in phase contrast microscopy at 40X magnification. Bars = 10 μm. 

The most notorious morphological changes were evident after the fungus was exposed to concentrations greater than 8 mM of KHCO₃. At 10 mM, the presence of primordia was noticed although not very well developed. Despite this, it was possible to observe the third structures of development (Figure 3, F), but it was not similar to the control by showing irregular formations of the sclerotium in which hyphal clusters were more loose (Figure 1, G-I). At 25 mM concentration, the formation of sclerotia primordia was scarce and disorganized (Figure 3, G-I), hence either the second or third structures of the sclerotium initiation could not be completed nor observed (Table 1). Finally, at the 50 mM concentration, no effects were noticed due to the absence of fungal growth in this treatment.

In this study, we observed the formation process of sclerotia during their initiation phase in which the four stages of development were identified (^{Bolton et al., 2006}; ^{Saharan and Mehta, 2008}). However, the sclerotial formation showed variations depending on the concentration of KHCO₃. The inhibitory effect of bicarbonates on the growth of several species of phytopathogenic fungi, especially during postharvest, has been recorded before (^{Aharoni et al., 1997}; ^{Palmer et al., 1997}; ^{Bombelli and Wright, 2006}; ^{Jabnoun-Khiareddine et al., 2016}), and ^{Ordóñez-Valencia et al. (2009)} demonstrated that KHCO₃ inhibited the growth of S. sclerotiorum in in vitro cultures. The inhibitory effect of bicarbonate salts on fungi was probably due to reduced fungal cell turgor pressure, which resulted in collapse and shrinkage of hyphae (^{Türkkan et al., 2017}).

The formation of sclerotial primordia by S. sclerotiorum was initiated by branching and clustering of hyphae (Figure 1), resulting in a mass of cells that eventually originate mature sclerotia. Similar effects were observed by ^{Smits and Noguera (1988)} in the formation of sclerotia of Macrophomina phaseolina, which began from hyphal branching and entwinements, besides the increase in size of the associate cells and the reduction in size of the sclerotial mass. ^{Townsend and Willets (1954)} observed different development patterns (thickening, branching, and septation of main hyphae and their entwinement) in Rhizoctonia solani, Botrytis allii, B. cinerea, and Sclerotium cepivorum.

In the present study, KHCO₃ resulted in microscopic morphological changes during early phases of sclerotia development. The increase of bicarbonate concentrations resulted in less profuse and loose hyphal branching, leading to the decrement and consequent absence of well-formed sclerotia (Table 1). ^{Igwegbe et al. (1977)} reported that the addition of 50 µg mL^-1 of 6-metilpurine caused significant reduction in the sclerotia formation by S. rolfsii.

The addition of KHCO₃ caused an increase in pH (from 6.5 to 8.0) in the culture medium (^{Ordóñez-Valencia et al., 2009}), which resulted in reductions of fungal growth. In this regard, ^{Alexander (1977)} mentioned that many fungi grow better under acidic conditions than alkaline, because an acidic environment is not conducive to the existence of either bacteria or actinomycetes, resulting in the monopoly of fungi for utilization of organic substrates (^{Giri et al., 2005}). On the other hand, it has been observed that both growth and development of sclerotia of S. sclerotiorum depend on the pH and the production of oxalic acid (^{Rollins and Dickman, 2001}; ^{Chen et al., 2004}). Neutral or alkaline pH values inhibit the formation of sclerotia, and the production of oxalic acid helps reducing the alkaline pH of the medium, creating more favorable conditions for the development of sclerotia (^{Rollins and Dickman, 2001}).

Although some reports have described the negative effects of bicarbonate on certain plant pathogenic fungi (^{Bombelli and Wright, 2006}; ^{Ordóñez-Valencia et al., 2009}), yet the present study is one of the first reports describing inhibitory effects of KHCO₃ on S. sclerotiorum during the initial phases of the sclerotia formation as well as on the morphology of sclerotial primordia.

The inhibitory effects of KHCO₃ on fungal growth and development may in part be explained by affecting vital biochemical processes such as the biogenesis of either the fungal cell wall and/or the apical wall (^{Sentandreu et al., 1994}; ^{Sideri and Georgiou, 2000}). Certain antimicrobial compounds cause oxidative stress in fungi which may show morphological changes, impaired growth rate, and low content of proteins and ATP (^{Harel et al., 2005}; ^{Marcet-Houben and Gabaldon, 2011}). In this regard, the application of KHCO₃ may account on the production of reactive oxygen species (ROS) as a response of the stress generated by this salt, then, causing alterations on the morphology and development of S. sclerotiorum.

Bicarbonate ions cause alterations in oxidation and nitration reactions in biological systems, regulate pH, and stimulate the production of either reactive nitrogen species such as peroxynitrite (ONOO-) or superoxide (O₂^-) (^{Knorev et al., 2000}; ^{Arai et al., 2005}; ^{Lushchak et al., 2009}). As a result of oxidative stress in combination with abiotic factors also have negative effects on the sclerotia formation in filamentous fungi (^{Georgiou et al., 2006}). Moreover, ^{Sideri and Georgiou (2000)} demonstrated that the production of hydrogen peroxide (H₂O₂) in S. rolfsii (Typhulaceae) exposed to different light and iron conditions; the highest production of H₂O₂ was recorded during early stages of fungal growth. However, as sclerotia become mature the hydrogen peroxide production decreased. Nevertheless, further research is needed to elucidate the effects of KHCO₃ on either physiological, biochemical or molecular processes during fungal morphogenesis.

CONCLUSIONS

The present results clearly demonstrate that KHCO₃ is effective in controlling both growth and development of S. sclerotiorum under in vitro conditions, which would help reducing the formation of sclerotia, as well as being a valid alternative for replacing or reducing the use ofiguraf synthetic fungicides. However, this is a preliminary study; thus, the efficiency of KHCO₃ must also be studied in a soil including plant-pathogen interactions. Moreover, S. sclerotiorum can spend 90% of its life cycle as sclerotia, thus it is important to direct more research efforts to know in detail the processes involved in either early initiation or further development of this persistent fungal structures.