The impact of 3 tested rhizobacterial strains (B. subtilis, B. polymyxa, and B. megaterium), Eugenol, and A. cina leaves extract on patho-physiological changes of wheat plants under stem rust stress was studied under laboratory and greenhouse conditions.
Microscope examination
Effect of the tested treatments on spore germination and disease symptoms
The effect of whole culture of the 3 Bacillus strains, Eugenol, and A. cina extract on spore germination of P. graminis on water agar medium was shown in Fig. 1. B. subtilis and Eugenol treatments had a higher impact for inhibition of spore germination than the control (Fig. 1b, f). On the other hand, spores treated with B. megaterium showed the highest germination percentage (Fig. 1d). Microscopic examinations of urediniospores exhibited abnormalities, lysis, collapse, and shrinking as a direct effect of the B. subtilis (b), B. polymyxa (c), B. megaterium (d), Eugenol (f), and Artemisia extract 2% (e) compared to (a) the control treatment (Fig. 2). It is well known that some Bacillus species produce extracellular metabolites such as lipopeptides and peptides with antibiotic activity and could be used as antagonists or growth promoters (Romero et al. 2008). Swelling and enlargement of mycelium were reported using lipopeptides by modification of the fungal membrane permeability and lipid composition.
The inhibiting effect on spore germination of stem rust pathogen is mainly due to antibiosis. Bacillus spp. produce at least 66 different antibiotic components (Ferreira et al. 1991). These antifungal materials inhibited growth of the pathogenic fungi and consequently reduced the disease. B. subtilis inhibited the growth of a wide range of fungal species including those in the Ascomycetes and Deuteromycetes (Qiao 2006). Likewise, E1R-j strain of B. subtilis inhibited urediniospores germination and germ tubes of P. striiformis in wheat (Li et al. 2013). The mycelial growth of Botrytis cinerea and Sclerotinia sclerotiorum were sensitive to Eugenol (Wang et al. 2010). Also, the inhibition of P. triticina urediniospores germination in vitro was achieved by using some plant extracts, i.e., Jacaranda mimosifolia, Thevetia peruviana, and Calotropis procera (Naz et al. 2014), while the selected treatments, i.e., Art. 2% + Zn, Art. 2% + Mn, Art. 2% + Fe, Art. 2% + Cu, Art. 1% + 50% Sumi-8, and Art. 1%, prevented spore germination of P. triticina (Omara et al. 2015). These results are in agreement with the obtained results, which clearly showed that the Bacillus strains, Eugenol, and A. cina extract significantly suppressed spore germinations of P. graminis on water agar medium with abnormalities and malformation of urediniospores.
Greenhouse studies
Effect of the tested treatments on incubation, latent periods, and infection type
The effects of the 3 bioagents and the 2 natural materials on stem rust development, such as incubation, latent periods, and infection type, were shown in Figs. 3 and 4. Different treatments gave significant differences when sprayed 24 h before and 24 h before and after inoculation of all parameters at 7 days from planting. Incubation and latent periods were increased in B. subtilis and Eugenol treatments. Besides, both treatments achieved the lowest infection type compared to the control. These results might be due to that Bacillus spp. produce several antibiotic compounds that are responsible of these changes in stem rust parameters (Ferreira et al. 1991). On the other hand, the incubation and latent periods as well as the infection type increased slowly in the B. megaterium treatment than the control treatment (Figs. 3 and 4). Likewise, the application of B. subtilis, T. viride, and plant extracts significantly increased both incubation and latent periods of leaf rust, caused by P. triticina f. sp. tritici (Omara et al. 2015 and 2019). Moreover, a foliar spray application at 24 h before and after inoculation was the best application on the seedling disease parameters, incubation and latent periods and infection type compared to application 24 h before inoculation.
The positive effects on both incubation and latent periods might be due to the induction of systemic resistance (ISR) as a main mechanism of plant response (Loeffler et al. 1986). Furthermore, the application of bioagents could positively alter the physical and mechanical strength of cell walls and adjust the physiological and biochemical responses of wheat plants, which can enhance the biosynthesis of defense-associated molecules that play a key role in delaying the inoculation and latent period of P. graminis. Additionally, Bacillus spp. have numerous approaches to control the disease such as the production of antifungal compounds, nutrient competition, and the induction of systemic resistance (Urszula et al. 2004).
Effect of the tested treatments on pustule size and receptivity (no. of pustules) of P. graminis at seedling stage
Figure 5 shows the reduction in pustule length, pustule width, and receptivity of P. graminis in different treatments. Both B. subtilis and Eugenol gave the lowest pustule length, pustule width, and receptivity than the control (untreated). In contrast, the B. megaterium treatment showed the lowest reduction in pustule length, pustule width, and receptivity. These results could be due to the production of antibiotics by B. subtilis such as bacillin, bacillomycin, bacitracin, mycosubtilin, subenolin, subsporin, and subtilin (Loeffler et al. 1986) reducing the development of the disease and pustule size. In addition, the membrane binding and permeability alteration were reported in Eugenol treatments leading to destabilization and disruption of the plasma membrane (Wang et al. 2010).
Laboratory studies
Antioxidant defense enzymes and electrolyte leakage
The activities of antioxidant defense enzymes including catalase (CAT), peroxidase (POX), and polyphenol oxidase (PPO) were displayed in Fig. 6a–c. Bacillus subtilis and Eugenol treatments resulted in a higher activity of CAT, POX, and PPO than control plants at 24, 72, and 168 h, but the highest increase was recorded at 72 h. These enzymes may participate in the responding defense reaction by induction of plant resistance against the pathogenic agents (Ray et al. 1998). Likewise, utilization of Bacillus spp. stimulated the enzymatic activities of chitinase, POX, and PPO against Rhizoctonia damping-off of snap bean plants (Ahmed 2016).
Catalase (CAT) as one of the oxidative enzymes plays an important role for increasing host resistance to control plant pathogens (Liau and Lin 2008). The increased activity of catalase prevented the increase of cytosolic hydrogen peroxide that creates toxic conditions leading to preventing pathogen spread and acting as a secondary signal for defense gene expression and activation of systemic acquired resistance (Neamat et al. 2016). Peroxidase is the first enzyme exhibiting changes in its activity under environmental stress (Milavec et al. 2001). Moreover, the changes in this antioxidant enzyme are known to be directly involved in the activation of plant defense responses. POX is known to catalyze the final polymerization step of lignin synthesis and is directly associated with the increased ability of systemically protected tissues for lignification (Chittoor et al. 1999). Therefore, the peroxidase activity could be used as a good indicator for plant resistance to diseases (Hameed et al. 2011). Also, PPO plays an important role in plant defense via the oxidation of endogenous phenolic compounds to the toxic quinines, which are toxic to the invading pathogens (Thakker et al. 2011). It may also participate in the inducible defense reaction and hypersensitivity for inducing resistance of plants to fungi, viruses, and bacteria (Hanifei et al. 2013).
As for electrolyte leakage, all treatments resulted in a clear reduction in electrolyte leakage in wheat plants infected with stem rust compared to control plants (Fig. 6d). These results may be due to the effect of the pathogen on the plasma membrane in control plants that increases the permeability of the plasma membrane. B. subtilis and Eugenol treatments were the best in decreased electrolyte leakage compared to the untreated plants. Plant stresses such as salinity, pathogen attack, drought, heavy metals, hyperthermia, and hypothermia accompanies electrolyte leakage (EL) as a mechanism of defense response.
Electrolyte leakage (EL) is an indication of increased permeability of the plasma membrane in the control plants than other treatments (Fig. 6d). The plasma membrane in the treatments was not affected by the infection, and its permeability was not increased (Omara et al. 2015). EL is related to K+ efflux from plant cells, which mediated by plasma membrane action conductance (Demidchik et al. 2014). The results were consistent with those obtained by Omara and Abdelaal (2018).
Characterization of B. subtilis metabolites by GC-MS analysis
Due to the performance of B. subtilis compared with other evaluated bioagents, MEMs (Micro-Electro Mechanical Systems) GC-MS technology was used to identify its active compounds. The most peak numbers and peak areas of compounds excreted by B. subtilis belonged to ten compounds. These compounds mostly included fatty acids known as 9-octadecanoic acid, octadecanoic acid, oleic acid, n-hexadecanoic acid, and hexadecanoic acid (Fig. 7).
Generally, numerous members of Bacillus species are known as producers of lipopeptides belonging to the surfactin, iturin, and fengycin families (Zuber et al. 1993). Fengycin is an antifungal lipopeptide complex produced by B. subtilis F-29-3 (Vanittanakom and Loeffler 1986). It consists of 2 main components, fengycin A and fengycin B. The lipid moiety of both analogs was more variable as fatty acids and identified as anteiso-pentadecanoic acid (ai-C15), iso-hexadecanoic acid (i-C16), and n-hexadecanoic acid (n-C16). There is an evidence for further saturated and unsaturated residues up to C18. In the present study, these components of fatty acids were detected in supernatant analysis, using GC-MS in case of B. subtilis. These results were consistent with those of Belal et al. (2013) who found that the methanolic extract of B. subtilis contained fatty acids such as nonanoic acid, decanoic acid, dodecatrienoic acid, heptadecanoic acid, octadecanoic acid, pentadecanoic acid, hexadecanoic acid, and some of their derivatives (Belal et al. 2013).
Principal component analysis (PCA)
Principal component analysis was performed on incubation and latent periods, infection type, pustule length, pustule width, receptivity (no. of pustule), catalase (CAT), peroxidase (POX), polyphenoloxidase (PPO), and electrolyte leakage in response to artificial infection of wheat stem rust and treatment with three Bacillus strains, Eugenol, and A. cina extract (Fig. 8a, b). Interpretation of the principal components (PCs) was aided by inspection of the factor-loading matrix extracted from a varimax rotation with Kaiser normalization of main components to identify factors responsible for the grouping of the dataset. As shown from scree plot graph of eigenvalues and loadings plot (Fig. 8), these 2 components, incubation and latent periods, have eigenvalues more than one. Therefore, these 2 components were extracted and describing approximately 96.595% of total variation; the remaining was 3.405% distributed on 8 factors. So, the variation of these factors is very small and negligible. Accordingly, principal component analysis gave an evidence to the importance of incubation and latent periods, as they were considered a good and more reliable indicator for evaluation of these materials. Summing up the results, the factor-loading matrix, extracted from a varimax rotation with Kaiser normalization, gave clear evidence to the importance of all parameters in this study, especially incubation and latent periods. Similar results were previously obtained when correlation statistics were performed between different disease parameters of wheat rusts and grain yield of the studied wheat genotypes (Abu Aly et al. 2017).