Plants use advanced strategies to defend themselves against biotic and abiotic stresses. Such basic resistance protects plants when facing pathogen attacks and helps them to reduce destructive effects of diseases via application of various biochemical, cellular and molecular defense mechanisms (Nikraftar et al, 2011). This means that plant simply doesn't provide the suitable environment for pathogens to attack.
In this study, the highest level of disease progress was observed on tomato seedlings and leaf discs infected with R. solani AG 4 HG-II. Infection caused by the binucleate isolate was less than multinucleate R. solani isolates. Generally, binucleate isolates of Rhizoctonia are reported as hypovirulent pathogens on various host plants, which in some cases have potential capability of controlling highly virulent multinucleate isolates of R. solani (Keshavarz-Tohid and Taheri, 2015; Sharon, et al., 2011). Therefore, it is interesting to find out if the hypovirulent binucleate isolate of Rhizoctonia used in this study is capable of inducing defense responses and protecting tomato plants against the highly virulent R. solani isolates, which can be the subject of future researches in this pathosystem.
Performing microscopic investigations of infection process in tomato-Rhizoctonia spp. interaction revealed the existence of direct correlation between the number of infection structures formed by the fungal isolates and disease progress on the leaves. The highest number of lobate appressoria and infection cushions was produced by AG 4 HG-II isolate, which caused the highest level of disease progress on tomato seedlings and leaf discs. Whereas, the lowest number of these infection structures was produced by the hypovirulent isolate of binucleate Rhizoctonia. These findings are in agreement with a previous report comparing the infection structures formed by R. solani AG 3 on a partially resistant (Falat) compared to a susceptible tomato cultivar (Mobile) for determining the correlation of infection structures formed by the pathogen with disease progress (Nikraftar et al. 2013). Infection cushions produced by R. solani AG 3 in Mobil cultivar at 48 hpi were more than of those of Falat cultivar (Nikraftar et al. 2013). Production of this infection structures in turn lead to the emergence of disease symptoms at the junction spot on the plants. Pannecoucque and Höfte (2009) stated that there is a difference between invasion activity and production of infection structures by various taxonomic groups of Rhizoctonia on cauliflower, which is in accordance with the findings of present study in tomato-Rhizoctonia pathostystem. Formation of infection structures also depends on plant cultivar and the site of pathogen attack. Verma (1996) explains that infection cushions are formed on hypocotyl of both resistant and susceptible plants but they spread faster and grow deeper in susceptible host plants. Bassi et al (1978) investigated resistant and one susceptible tomato cultivars to Rhizoctonia and observed that the pathogen spreads in the resistant cultivar much slower than in the susceptible one. Thus, inability of Rhizoctonia to form a lot of infection cushions in the partially resistant cultivar could be associated with lower level of disease development on it.
One of the most important types of ROS is H2O2, which is known to be involved in tomato resistance against biotrophic fungal pathogens such as Cladosporium fulvum (Borden and Higgins, 2002), Oidium neolycopersici (Mlikova et al, 2004), and hemibiotrophics such as Colletotrichum coccodes (Mellersh et al, 2002). In addition, several studies revealed the importance of H2O2 cumulated in tomato as a defense response against necrotrophic fungi such as Botrytis cinerea (Asselbergh, 2007), Fusarium oxysporum f.sp. lycopersici (Mandal et al, 2008)d solani (Nikraftar et al, 2013). The results of this study revealed that the highest amounts of not only H2O2, but also O2− were produced in the leaf tissues of tomato plants infected with R. solani AG 3, while the lowest levels of these molecules were produced in plant cells interaction with the hypovirulent isolate of binucleate Rhizoctonia. Therefore, it can be concluded that oxidative burst is one of host defense responses against aggressive isolates of R. solani. Accumulation of higher amount of ROS is correlated with lower levels of disease progress and higher resistance of the host plant in tomato-R. solani interaction.
Callose is a 1,3- β glucan polymer found in plants, which is produced in response to lesions, biotic or abiotic stresses (Usak et al. 2023; Li et al. 2023). In the present research, it was observed that inoculating tomato plants with highly virulent R. solani isolates (belonging to AG 4 HG-I and AG 4 HG-II) led to accumulation of lower levels of callose compared to the amount of callose accumulated in plants infected with the isolate of AG 3, with lower level of virulence. Therefore, virulence of R. solani isolates was inversely related to callose deposition in the host cells. In other words, higher level of tomato resistance to R. solani AG 3 compared to AG 4 isolates was correlated with higher amounts of callose deposition. These data suggest that callose deposition can be considered as a resistance marker in tomato-R. solani pathosystem. This finding is in agreement with the report of Gindro et al (2006) in grape-Plasmopara viticola interaction, which stated that the percentage of infected stoma representing callose deposition can be used as a marker to examine resistance of various grape varieties to downy mildew. Researchers found that callose is deposited with more delay in mesophyll of susceptible grape varieties compared to the resistant ones. By forming papillae, callose deposition prevents penetration of haustoria formed by phytopathogenic fungi into the epidermal cells of host plants (Heinz et al, 1990), therefore it can be a powerful physical barrier for penetration of phytopathogens into the plant cells. Romero et al. (2008) demonstrated that oxidative burst and strengthening of plant cell wall through accumulation of callose and lignin in melon varieties resistant to powdery mildew, caused by Podosphaera fusca, is more than those of susceptible varieties, which is in accordance with our findings.
Employment of X/XO leads to production of O2− and H2O2, resulting in increased disease progress by R. solani, similar to the findings of other researchers for necrotophic pathogens such as Botrytis cinerea (Govrin and Levin, 2000) and Cochliobolus miyabeanus (De Vleesschauwer, 2009). On the other hand, G/GO treatment increases H2O2 accumulation in plant tissues (Bennett et al, 2005; Mur et al, 2005) that can be associated with activation of defense mechanisms leading to decrease disease symptoms. Analysis of defense genes induced by lesions in tomatoes revealed that H2O2 functions as a secondary messenger and plays an important role in induction of defense genes (Orozco-Cárdenas et al, 2001). The results of this study also indicated that increased amount of H2O2 by G/GO results in a significant reduction of disease severity. It is assumed that H2O2 produced over the interaction between plant and the necrotophic fungus R. solani functions as a secondary messenger that induces defense genes just like in lesions. According to the result of evaluating callose deposition in the leaf discs treated with G/GO and X/XO and inoculated with different taxonomic groups of the pathogen, callose accumulation in the G/GO treated leaves was higher compared to X/XO treated leaves. Therefore, it can be concluded that lower levels of disease progress in G/GO treated leaved is correlated with higher amount of callose deposition via this treatment.
Ascorbic acid (ascorbate) is a potent antioxidant, which is involved in removing H2O2 from living cells. The findings of this study revealed that accumulation of H2O2 is of great importance in basal resistance of tomato to different taxonomic groups of Rhizoctonia, as susceptibility of the host plant to these fungi increased via treating leaf discs with ascorbate. Asselbergh et al (2007) also reported that tomato leaf discs treated with ascorbate showed increased susceptibility to the necrotrophic fungus B. cinerea. Ascorbate treatment, on the other hand, leads to unwanted changes in the natural metabolism of plant cells. The reason is that ascorbate is used as an electron donor in reduction reactions of biological processes (Taheri 2022; Noctor, 2006).
Investigating activity of the antioxidant enzymes, such as the SOD and POX, which are involved in regulating ROS levels, revealed higher level of SOD activity for the plants with the hypovirulent BNR and R. solani AG-3 treatments. This observation was in agreement with the data obtained in transcription analyses of the corresponding gene. Considering the function of SOD, which converts O2− into H2O2 and increase H2O2 accumulation, like the G/GO treatment which produces H2O2 and leads to decreased disease progress, the data obtained in enzyme activity, gene expression and ROS manipulations assay are in accordance. As we observed that the R. solani AG 3 isolate, which caused lower levels of the disease symptoms compared to other isolates of R. solani tested, showed the highest SOD activity and higher amounts of H2O2 accumulation at the early time point investigated (12 hpi). H2O2 is known as a second messenger involved in plant defense against R. solani (Kheyri et al. 2022, Nikraftar et al. 2014) and also may be involved in production of callose and lignin as the main physical barriers which prevent progress of the fungal pathogen in the plant tissues (Taheri 2022).
The POX activity and upregulation of the corresponding gene, which was higher in the early time point 12 hpi in the plants inoculated with R. solani AG 4 HG-I and AG 4 HG-II, might be related to the role of this antioxidant in degrading H2O2 as a defense related signaling molecule, which lead to higher level of the disease progress on the plants inoculated with these taxonomic groups of R. solani. These findings are in accordance with the results obtained by Kheyri et al. (2022) about detecting lower levels of H2O2 in the plants without the protectant treatment that had higher disease progress, compared to the plants with resistance inducer treatments which showed higher H2O2 accumulation at some early time points.
Plant cell wall is composed of various polysaccharides and several proteins. In all cases, polysaccharides (such as cellulose) constitute a major part of plant cell walls and pectin can be found in middle lamella. The cell polysaccharides are classified into cellulose, hemicellulose and pectin and these three can be found in almost all plant cell walls in different proportions (Harholt et al, 2010; Harholt et al, 2006). To destroy the cell wall, exocellular proteins such as cellulolytic, hemicellulolytic, pectolytic and proteolytic enzymes are produced by phytopathogens, which are able to attack cell wall components (Viler, 1975). Therefore, pathogens can produce and secret a wide range of enzymes capable of degrading plant cell wall ingredients, which are necessary for successful penetration into the host cells (Jayasinghe et al. 2004; Khaledi et al. 2015). Findings of this study clarified presence of a direct correlation between pathogenicity of Rhizoctonia isolates and activity of cellulase and pectinase enzymes produced by them. However, it seems necessary to make further investigations on other taxonomic groups of this fungus and use several isolates of each taxonomic group to survey the relationship between activity of cell wall degrading enzymes secreted by pathogens and their pathogenicity on the host plant.
In accordance with our data, Pannecoucque and Höfte (2009) demonstrated that during the pathogenic interactions between Rhizoctonia isolates and cauliflower, pectin degrading enzymes are important and diffused ahead of the fungus and pathogen ingress is coupled with host cell deformation and pectin lysis at locations not in direct contact with fungal hyphae. For several phytopathogenic fungi, including Rhizoctonia, the role of cellulase and pectin degrading enzymes in penetrating plant tissues, virulence and aggressiveness of the pathogen is demonstrated (Jayasinghe et al. 2004; Xue et al. 2018). Bateman (1964) reported that R. solani produces cellulase which may assist penetration of the fungus into host cells. Pectinase activity was regarded to be the most important predictor for virulence of R. solani (Weinhold and Bowman 1974; Mandal et al. 2013). Degradation of pectin in middle lamella and cellulose contents of the cell wall plays a considerable arole in development of pathogen in plant tissue and providing nutriments for it. Therefore, cellulase and pectinase are potentially important for aggressiveness and virulence of various phytopathogens. Investigating their activity together with the activity of other CWDEs for several isolates from each taxonomic group may be used as helpful biochemical markers for estimating pathogenicity of numerous fungal isolates, without performing time-consuming greenhouse trials.