Revista mexicana de fitopatología
versão On-line ISSN 2007-8080versão impressa ISSN 0185-3309
Rev. mex. fitopatol vol.36 no.3 Texcoco Out./Dez. 2018
https://doi.michael-shanks.com/10.18781/r.mex.fit.1803-4Early morphological development of sclerotia of Sclerotinia sclerotiorum in the presence of potassium bicarbonate
Claudia Ordóñez-Valencia1
Ronald Ferrera-Cerrato1
Alejandro Alarcón1 *
Laura V. Hernández-Cuevas2
John Larsen3
1 Área de Microbiología, Posgravày de Edafología.Colegio de Postgraduados. Carretera México-Texcoteo Km 36.5. Montecillo, Texcoteo,Estavị de Méxiteo, CP.. 56230 México
2 Centro de Investigación en Genética yAmbiente. Universidad Autónoma de Tlaxcala. Autopista Texmelucan-Tlaxcala Km10.5. Ixtacuixtla CP.. 901đôi mươi, Tlaxcala, México
3 Instituto 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, CPhường. 58190 México.
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Abstract:
Sclerotinia sclerotiorum is a pathogene 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 in the soil for many years. This study evaluated the morphological development of S. sclerotiorum sclerotium primordia by using the Riddell technique, & 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 và inhibits the development of sclerotia in their initial stages; hence it may offer potential as an alternative to lớn synthetic fungicides for the control of plant diseases caused by S. sclerotiorum.
Key words: antifungal agent; morphogenesis; inhibition; microscopy
Resumen:
Sclerotinia sclerotiorum es un patógeno de suma importancia económica quecausa grandes pérdidas en varios cultivos. Controlar este patógeno es difícilporque forma estructuras de resistencia llamadas esclerocgame ios que puedenmantenerse viables en el suelo por muchos años. Este estudio evaluó eldesarrollo morfológiteo de los primordios de escleroctiện ích ios de S.sclerotiorum utilizanvị la técnica de Riddell, y diferentesconcentraciones de bicarbonato lớn 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 escleroctiện ích ios, así como en lainhibición de su desarrollo al utilizar umãng cầu concentración de 50 mM deKHCO3. Este compueskhổng lồ químiteo modifica la morfología e inhibe eldesarrollo de escleroctiện ích ios en su fase inicial y, por tanlớn, 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 lớn the family Sclerotiniaceae. This is a destructive fungal pathoren for many agricultural crops such as sunflower, soybean, oilseed rape, bean, chickpea, canola, and onion (Hegedus và Rimmer, 2005: Bolton et al; 2006). It has little host specifithành phố, thus being able to infect over 400 plant species, mainly dicotyledons (Fernanvì chưng et al., 2004; Hegedus and Rimmer, 2005). The environmental conditions that promote the fungal infection are high humidity & temperatures between 15 & 25 °C (Saharan and Mehta, 2008). Secretion of fungal enzymes such as cellulases and pectinases, that soften & degrade plant tissues are involved in the plant infection process (Fernando 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 and Rimmer, 2005). One characteristic of this pathogene is the formation of sclerotia, fungal structures of resistance và dispersal, which under favorable conditions can remain viable for several years in soils (Bae and Knudsen, 2007; Calvo and Cary 2015; Smith et al., 2015).
During the formation of sclerotia, three stages or phases have been identified: 1) initiation: aggregation of hyphae, 2) development: hyphal growth for greater kích cỡ, và 3) maturation: creation of surface boundaries, internal consolidation and melanization (Le Tourneau, 1979; Rollins and Dickman, 2001; Bolton et al., 2006; Saharan & Mehta, 2008). The initiation and maturation stages may be influenced by abiotic factors such as photoperiod, temperature, oxygene, and nutrient availability (e.g. carbon sources), và the morphogenesis & further development of sclerotia started between 12 and 24 h of fungal growth (Hansberg và Aguirre, 1990).
The sclerotium is composed by three layers: a thiông xã & pigmented outer layer, an intermediate & thin layer, & an internal trắng layer called the inner medulla (Punja and Damiani, 1996; Bardin và Huang, 2001). Depending on environmental conditions, sclerotia grow belowground in one of two ways: 1) by forming mycelium that potentially infects roots and causes rot và wilting of plant tissues, or 2) by producing apothecia, in which ascospores are produced & released, then infecting aerial plant michael-shanks.comans (Humpherson-Jones và Cooke, 1977; Mónaco et al., 1998; Bolton et al., 2006).
Bicarbonates possess antimicrobial properties of wide spectrum, & their efficiency has been proven for controlling many plant pathogenic fungi (Bombelli & Wright, 2006; Arslan, 2015). The Environmental Protection Agency (EPA) of the United States also recognizes bicarbonates as innocuous & safe compounds for both human health & environment (Palmer et al., 1997; Bombelli & Wright, 2006), since their use may decrease the utilization of pesticides. Some studies have sầu shown that sodium, potassium & ammonium carbonates và bicarbonates inhibit the post-harvest growth of several fungal pathogens in fruits, vegetables & 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 sầu phosphorylation, và exert toxic effects on the structures of the pathogene (Avis, 2007). However, their efficacy depends on the concentration (0.2-3%) và on the susceptibility of each micromichael-shanks.comanism. For instance, treatments with sodium carbonate & bicarbonate improved the control of the green mold caused by Penicillium digitatum Sacc. (Trichocomaceae), in citrus fruits (Smilanichồng et al., 1999). Sodium and potassium bicarbonates also reduced powdery mildew caused by Leveillula taurica (Lév.) Arnaud (Erysiphaceae) in peppers (Fallik et al., 1997), & decreased the conidiogenesis by Helminthosporium solani Durieu y Mont. (Pleosporaceae) (Olivier et al, 1998). In addition, Bombelli & Wright (2006), & Türkkan et al. (2017) observed the growth inhibition of Botrytis cinerea Pers. Fr. (Sclerotiniaceae) when exposed khổng lồ different bicarbonates with in vitro cultures. Plant disease control in carrot, cucumber và cantaloupe fruits has been also reported due to lớn the application of bicarbonates (Aharoni et al., 1997; Bombelli và 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 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 ayên of this study was to evaluate the effects of different doses of potassium bicarbonate on the early stages và primordia development of sclerotia by S. sclerotiorum via microscopic observations.
MATERIALS AND METHODS
In order lớn 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) và 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 khổng lồ 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 cốt tông blue in lactophenol was added. The next step was khổng lồ 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, & 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).
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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, và 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) và 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 & sclerotia (Figure 2 and 3). However, at 2 mM, 4 mM and 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 (đưa ra square “goodness fit”).
| Tratamientos Concentraciones de bicarbonalớn de sodio | ||||||||
| Fase de la estructura | Control | 2 mM | 4 mM | 6 mM | 8 mM | 10 mM | 25 mM | Probabilidad |
| I | 6 | 5 | 5 | 6 | 5 | 5 | 7 | 0.8606 |
| II | 5 | 5 | 5 | 5 | 5 | 5 | 0 | 0.2914 |
| III | 4 | 5 | 5 | 6 | 5 | 5 | 0 |
Figure 2 Microscopic developmental structures observed during the sclerotial formation of Sclerotinia sclerotiorum, after seven days of fungal growth. First (indicated by arrows), second, và 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, & (D-F) 10 mM. For 25 mM a cellular dismichael-shanks.comanization of the primordium was observed (G-I), in which the second và 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 lớn observe sầu the third structures of development (Figure 3, F), but it was not similar 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 & dismichael-shanks.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 lớn 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 and Wright, 2006; Jabnoun-Khiareddine et al., 2016), & 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 khổng lồ 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 và Noguera (1988) in the formation of sclerotia of Macrophomina phaseolina, which began from hyphal branching and entwinements, besides the increase in kích thước of the associate cells & the reduction in kích thước of the sclerotial mass. Townskết thúc & Willets (1954) observed different development patterns (thickening, branching, & septation of main hyphae và 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 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 khổng lồ the existence of either bacteria or actinomycetes, resulting in the monopoly of fungi for utilization of michael-shanks.comanic substrates (Giri et al., 2005). On the other hand, it has been observed that both growth & development of sclerotia of S. sclerotiorum depover on the pH và the production of oxalic acid (Rollins & Dickman, 2001; Chen et al., 2004). Neutral or alkaline pH values inhibit the formation of sclerotia, và 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 sầu described the negative effects of bicarbonate on certain plant pathogenic fungi (Bombelli và 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 & 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 và Gemichael-shanks.comiou, 2000). Certain antimicrobial compounds cause oxidative bức xúc in fungi which may show morphological changes, impaired growth rate, & low content of proteins và ATPhường (Harel et al., 2005; Marcet-Houben & Gabaldon, 2011). In this regard, the application of KHCO3 may tài khoản on the production of reactive sầu oxygen species (ROS) as a response of the găng tay generated by this salternative text, then, causing alterations on the morphology & development of S. sclerotiorum.
Bicarbonate ions cause alterations in oxidation and nitration reactions in biologicalsystems, regulate pH, và stimulate the production of either reactive nitrogenspecies such as peroxynitrite (ONOO-) or superoxide (O2-)(Knorev et al.,2000; Arai et al.,2005; Lushchak etal., 2009). As a result of oxidative áp lực in combinationwith abiotic factors also have negative sầu effects on the sclerotia formation infilamentous fungi (Gemichael-shanks.comiou etal., 2006). Moreover, Sideri & Gemichael-shanks.comiou (2000) demonstrated that the production ofhydrogene peroxide (H2O2) in S. rolfsii(Typhulaceae) exposed lớn different light and 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.
