Revista mexicana de fitopatología
versão On-line ISSN 2007-8080versão impressa ISSN 0185-3309
Rev. mex. fitopatol vol.36 no.3 Texcoco Out./Dez. 2018https://doi.onfire-bg.com/10.18781/r.mex.fit.1803-4
Early morphological development of sclerotia of Sclerotinia sclerotiorum in the presence of potassium bicarbonate
Alejandro Alarcón1 *
Laura V. Hernández-Cuevas2
1 Área de Microbiología, Posgravị de Edafología.Colegio de Postgraduados. Carretera México-Texcoteo Km 36.5. Montecillo, Texcoteo,Estabởi vì de Méxiteo, CP. 56230 México
2 Centro de Investigación en Genética yAmbiente. Universidad Autónoma de Tlaxcala. Autopista Texmelucan-Tlaxcala Km10.5. Ixtacuixtla CPhường. 901trăng tròn, Tlaxcala, México
3 Institukhổng lồ de Investigaciones en Ecosistemas ySustentabilidad, Universidad Nacional Autónoma de México. Antigua Carretera aPátzcuaro No.8701, Colonia Ex Haciendomain authority de San José de la Huerta, Morelia,Michoacán, CP.. 58190 Méxiteo.
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Sclerotinia sclerotiorum is a pathoren of great economic importance that causes significant losses in various crops. Control of the pathogen is difficult since this fungus forms resistant sclerotia that can survive sầu in the soil for many years. This study evaluated the morphological development of S. sclerotiorum sclerotium primordia by using the Riddell technique, và different concentrations of potassium bicarbonate (KHCO3). The formation of primordia began from hyphae. However, as the concentration of KHCO3 increased, morphological changes were observed in the initiation phase of the sclerotia, as well as in the inhibition of their development when using a 50 mM concentration of KHCO3. This chemical compound modifies the morphology & inhibits the development of sclerotia in their initial stages; hence it may offer potential as an alternative khổng lồ synthetic fungicides for the control of plant diseases caused by S. sclerotiorum.
Key words: antifungal agent; morphogenesis; inhibition; microscopy
Sclerotinia sclerotiorum es un patógeno de suma importancia económica quecausa grandes pérdidas en vargame ios cultivos. Controlar este patógeno es difícilporque forma estructuras de resistencia llamadas esclerocquả táo que puedenmantenerse viables en el suelo por muchos años. Este estudio evaluó eldesarrollo morfológiteo de los primordgame ios de esclerocios de S.sclerotiorum utilizanvị la técnica de Riddell, y diferentesconcentraciones de bicarbonakhổng lồ de potasio (KHCO3). La formación delos primordtiện ích ios de los esclerocquả táo inició a partir de hifas; sin embargo,conforme las concentraciones de KHCO3 incrementaron, se observaroncambgame ios morfológicos en la fase de iniciación de los esclerocquả táo, así como en lainhibición de su desarrollo al utilizar una concentración de 50 mM deKHCO3. Este compuesto lớn químiteo modifica la morfología e inhibe eldesarrollo de escleroctiện ích ios en su fase inicial y, por tanto, podría utilizarsecomo alternativa a los fungicidas sintéticos para controlar enfermedades deplantas causadas por S. sclerotiorum.
Palabras clave: agente antifúngico; morfogénesis; inhibición; microscopia
White mold is caused by Sclerotinia sclerotiorum, a fungus that belongs to lớn the family Sclerotiniaceae. This is a destructive fungal pathogene for many agricultural crops such as sunflower, soybean, oilseed rape, bean, chickpea, canola, and onion (Hegedus & Rimmer, 2005: Bolton et al; 2006). It has little host specificity, thus being able to infect over 400 plant species, mainly dicotyledons (Fernanvày et al., 2004; Hegedus and Rimmer, 2005). The environmental conditions that promote the fungal infection are high humidity và temperatures between 15 & 25 °C (Saharan và Mehta, 2008). Secretion of fungal enzymes such as cellulases & pectinases, that soften và degrade plant tissues are involved in the plant infection process (Fernanvì chưng et al., 2004; Bolton et al., 2006), as well as production of oxalic acid, which has toxic effects on the tissue of the host (Hegedus và Rimmer, 2005). One characteristic of this pathogen is the formation of sclerotia, fungal structures of resistance và dispersal, which under favorable conditions can remain viable for several years in soils (Bae & Knudsen, 2007; Calvo và Cary 2015; Smith et al., 2015).
During the formation of sclerotia, three stages or phases have sầu been identified: 1) initiation: aggregation of hyphae, 2) development: hyphal growth for greater form size, and 3) maturation: creation of surface boundaries, internal consolidation & melanization (Le Tourneau, 1979; Rollins & Dickman, 2001; Bolton et al., 2006; Saharan & Mehta, 2008). The initiation & maturation stages may be influenced by abiotic factors such as photoperiod, temperature, oxygene, & nutrient availability (e.g. carbon sources), and the morphogenesis và further development of sclerotia started between 12 & 24 h of fungal growth (Hansberg & Aguirre, 1990).
The sclerotium is composed by three layers: a thick and pigmented outer layer, an intermediate and thin layer, & an internal White layer called the inner medulla (Punja và Damiani, 1996; Bardin and Huang, 2001). Depending on environmental conditions, sclerotia grow belowground in one of two ways: 1) by forming mycelium that potentially infects roots & causes rot và wilting of plant tissues, or 2) by producing apothecia, in which ascospores are produced & released, then infecting aerial plant onfire-bg.comans (Humpherson-Jones và Cooke, 1977; Mónateo et al., 1998; Bolton et al., 2006).
Bicarbonates possess antimicrobial properties of wide spectrum, and their efficiency has been proven for controlling many plant pathogenic fungi (Bombelli và Wright, 2006; Arslan, 2015). The Environmental Protection Agency (EPA) of the United States also recognizes bicarbonates as innocuous và safe compounds for both human health and environment (Palmer et al., 1997; Bombelli & Wright, 2006), since their use may decrease the utilization of pesticides. Some studies have shown that sodium, potassium và ammonium carbonates và bicarbonates inhibit the post-harvest growth of several fungal pathogens in fruits, vegetables and ornamental plants (Karabulut et al., 2003; Arslan et al., 2006; Jabnoun-Khiareddine et al., 2016). Bicarbonates alter the permeability of fungal membranes, inhibit the reactions of oxidative phosphorylation, and exert toxic effects on the structures of the pathogen (Avis, 2007). However, their efficacy depends on the concentration (0.2-3%) & on the susceptibility of each microonfire-bg.comanism. For instance, treatments with sodium carbonate & bicarbonate improved the control of the green mold caused by Penicillium digitatum Sacc. (Trichocomaceae), in citrus fruits (Smilaniông xã et al., 1999). Sodium and potassium bicarbonates also reduced powdery mildew caused by Leveillula taurica (Lév.) Arnaud (Erysiphaceae) in peppers (Fallik et al., 1997), and decreased the conidiogenesis by Helminthosporium solani Durieu y Mont. (Pleosporaceae) (Olivier et al, 1998). In addition, Bombelli và Wright (2006), & Türkkan et al. (2017) observed the growth inhibition of Botrytis cinerea Pers. Fr. (Sclerotiniaceae) when exposed lớn different bicarbonates with in vitro cultures. Plant disease control in carrot, cucumber và cantaloupe fruits has been also reported due lớn the application of bicarbonates (Aharoni et al., 1997; Bombelli and Wright, 2006).
Overall, the inhibitory effects of potassium bicarbonate (KHCO3) on the growth of S. sclerotiorum, as well as on the germination and formation of new sclerotia have sầu been described (Ordóñez-Valencia et al., 2009), but the effects of this chemical compound during early stages of sclerotia morphological development are not well understood. Thus, the aim of this study was lớn evaluate the effects of different doses of potassium bicarbonate on the early stages and primordia development of sclerotia by S. sclerotiorum via microscopic observations.
MATERIALS AND METHODS
In order khổng lồ maintain the humidity in the Petri dishes, 10 mL of 10% glycerol were added. All Petri dishes were kept at room temperature conditions (~trăng tròn °C) & 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 lớn prepare the fungal slides. The cover slip was carefully separated from the agar và placed on another clean slide on which a drop of the colorant cốt tông blue in lactophenol was added. The next step was khổng lồ remove sầu the PDA-disk from the original microscopic slide on which the colorant was also added, và 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, & 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 khổng lồ 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).
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RESULTS AND DISCUSSION
The presented fungal structures are part of the process of sclerotia formation by S. sclerotiorum, và 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, và 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 sầu 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) & 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 & less compact. The increase in the concentration of bicarbonate resulted in growth inhibition of both hyphae và sclerotia (Figure 2 and 3). However, at 2 mM, 4 mM và 6 mM concentrations of KHCO3 only scarce morphological changes were observed.
Table 1 Frequencies of sclerotia on each structure phase. The value on last column indicates the probability of not effects due to lớn sodium bicarbonate exposition for each structure phase (bỏ ra square “goodness fit”).
|Tratamientos Concentraciones de bicarbonakhổng lồ de sodio|
|Fase de la estructura||Control||2 mM||4 mM||6 mM||8 mM||10 mM||25 mM||Probabilidad|
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, & (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 và third structures of sclerotial development in presence of KHCO3: (A-C) 8 mM, and (D-F) 10 mM. For 25 mM a cellular disonfire-bg.comanization 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 khổng lồ 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 lớn observe the third structures of development (Figure 3, F), but it was not similar to lớn 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 & disonfire-bg.comanized (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 và 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 & Wright, 2006; Jabnoun-Khiareddine et al., 2016), và 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 lớn reduced fungal cell turgor pressure, which resulted in collapse và shrinkage of hyphae (Türkkan et al., 2017).
The formation of sclerotial primordia by S. sclerotiorum was initiated by branching & clustering of hyphae (Figure 1), resulting in a mass of cells that eventually originate mature sclerotia. Similar effects were observed by Smits & Noguera (1988) in the formation of sclerotia of Macrophomimãng cầu phaseolimãng cầu, which began from hyphal branching và entwinements, besides the increase in size of the associate cells và the reduction in form size of the sclerotial mass. Townskết thúc và Willets (1954) observed different development patterns (thickening, branching, and septation of main hyphae & their entwinement) in Rhizoctonia solani, Botrytis allii, B. cinerea, & 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 & loose hyphal branching, leading khổng lồ the decrement và 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 lớn 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 sầu to the existence of either bacteria or actinomycetes, resulting in the monopoly of fungi for utilization of onfire-bg.comanic substrates (Giri et al., 2005). On the other h&, it has been observed that both growth và development of sclerotia of S. sclerotiorum depover on the pH & the production of oxalic acid (Rollins và 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 & 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 và 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 & Geonfire-bg.comiou, 2000). Certain antimicrobial compounds cause oxidative sầu ức chế in fungi which may show morphological changes, impaired growth rate, & low content of proteins và ATP. (Harel et al., 2005; Marcet-Houben and Gabaldon, 2011). In this regard, the application of KHCO3 may trương mục on the production of reactive sầu oxygene species (ROS) as a response of the bức xúc generated by this salt, then, causing alterations on the morphology and development of S. sclerotiorum.
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Bicarbonate ions cause alterations in oxidation and nitration reactions in biologicalsystems, regulate pH, and stimulate the production of either reactive sầu nitrogenspecies such as peroxynitrite (ONOO-) or superoxide (O2-)(Knorev et al.,2000; Arai et al.,2005; Lushchak etal., 2009). As a result of oxidative sầu ức chế in combinationwith abiotic factors also have sầu negative sầu effects on the sclerotia formation infilamentous fungi (Geonfire-bg.comiou etal., 2006). Moreover, Sideri and Geonfire-bg.comiou (2000) demonstrated that the production ofhydroren peroxide (H2O2) in S. rolfsii(Typhulaceae) exposed to different light & iron conditions; the highestproduction of H2O2 was recorded during early stages offungal growth. However, as sclerotia become mature the hydroren peroxideproduction decreased. Nevertheless, further research is needed to elucidate theeffects of KHCO3 on either physiological, biochemical or molecularprocesses during fungal morphogenesis.
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