The total area for worldwide strawberry (
Under biotic and abiotic stresses, morpho-anatomical changes such as trichome increments are triggered in plants (Figueiredo et al., 2013; Lucini et al., 2015), increasing the thickness of the epidermis and foliar mesophyll, synthesis of crystal waxes, tissue resistance and functioning of substance secreting structures (Tenhaken, 2015; Zhu, 2016).
The synthesis of the volatile methyl salicylate compound (MeSa) is one of the main signs of a plant's response to stresses. Through the release of MeSa, the neighbouring plants are alerted by activating specific genes that are transcribed into proteins that assist in the synthesis of defence structures or substances (Backer et al., 2019; Miller et al., 2017). MeSa is derived from salicylic acid (SA) and is produced through secondary metabolism. SA is found in leaves and other plant parts, and its concentration inside different plant cells is varied (Hayat et al., 2012). Biochemical studies suggest that SA is synthesised from the phenylalanine pathway with benzoate as the immediate precursor (Hayat et al., 2014). However, more recent genetic analyses indicated that the bulk (>90%) of SA is synthesised from the isochorismate pathway (Chen et al., 2009). Although the role of plant isochorismate syntheses in SA production has been established, plant enzymes that convert isochorismate into SA have not yet been identified.
The application of exogenous SA in plants promotes increased resistance to pests and diseases that cause extensive damage to commercial crops. In addition, SA can replace/decrease the use of pesticides and/or reduce waste in the final product, increasing profitability and encouraging the approach toward sustainable agriculture (Donovan and Delucia, 2013; Wani et al., 2017). Exogenous SA also provides conditions for plants to overcome situations of abiotic stress, including heat, drought and saline environments (Nazar et al., 2017).
Exogenous SA has been reported to induce resistance to the striped mite in strawberry cultivars, reducing adult survival and oviposition (Favaro et al., 2019). However, the mechanisms related to these resistance effects are not yet fully understood, especially regarding the morpho-agronomic changes that occur in plants attacked by the pest. Thus, knowing these forms of defence that the plant develops can favour the search for more sustainable crops. Given the issues that are discussed above, the objective of this research was to evaluate the effects of exogenous SA on the development of morpho-anatomical defence structures in strawberry plants and the relationship of these structures with the biology and behaviour of
For the test, juvenile strawberry (‘Sweet Charlie’) plants obtained from the nursery were grown in pots (3 dm3 per 40 days) containing a mixture of soil and commercial bio-stabilised substrate, obtained by anaerobic digestion in a reactor and fertilised with 3.8 g NPK formula 4-14-8 (NPK) in an automated greenhouse (T 25 ± 3oC, RH 80 ± 5%, and photoperiod 13:11 h, light:dark). For each treatment, eight plants were taken and divided into four replications with two plants per pot, based on the preliminary research carried out by Favaro et al. (2019).
Application of SA was initiated 30 days after transplanting. Five concentrations (0 mg · L−1, 25 mg · L−1, 50 mg · L−1, 75 mg · L−1 and 100 mg · L−1) were applied using an electric sprayer of MTS brand, model Spritz 18®, with a capacity of 10 L, driven by an electric motor (with a power of 30 W) and pressure sensor of the differential type (precision of 2.5% and response time of 1 s). A nozzle model Xr 110.02 was used, with a flat spray with a pressure of 300 kPa, a target height of 0.5 m and a nominal flow of 0.78 L · min−1. Foliar surfaces were treated until saturation point (dripping). The plants were sprayed with SA at 15 days’ time intervals after transplantation, for 5 months. The plots were separated by 3 m and consisted of two pots with one plant from the pot. After the third application, 60 days after the transplant, 12 adult mites of the same age were released per plant in the proportion of 3 females for each male, in line with Favaro et al. (2019). The doses of SA used in the bioassay were also established by Favaro et al. (2019). Yet, in these trials, it was postulated that infestation with the pest was necessary for plants to respond with morphological changes on application of SA.
Expanded juvenile leaflets were sampled on the upper third of the plants, 45 days after infestation with TSSM. Two leaflets from the plant were removed from each replicate, totalling 16 samples per treatment. From each sample, two paradermic cuts were made on the abaxial and adaxial surfaces of the leaflet with a rotating microtome, totalling 32 cuts per treatment. The sampled cut leaflets were fixed with a metallic substrate using carbon tape (Goldstein et al., 1992). The images were captured by SEM (Tescan Vega3 HV 30kv) in an area of 300 μm2 of the leaflet. Trichomes were quantified according to the presence/absence of the glands at the apex of the trichome from 32 images, obtained at the best resolution. The values were determined for each type of trichome (glandular or non-glandular) on the surface of the leaflet (adaxial and abaxial) and weighted to obtain the average of each treatment.
For the transverse histological cuts, two leaflets were sampled from a plot (one per plant) collected from four replicates/treatment, totalling eight leaflets. From each leaflet, four paradermic cuts were made on the microtome rotary (MRP2015) with a thickness of 5 mm, totalling 32/treatment. Subsequently, 10 best cuts for capturing images under SEM (200 μm, 100 μm and 50 μm of resolution) were selected. Through image analysis, the thickness of the leaflet (LT), abaxial cell wall (AbCW), adaxial cell wall (AdCW), palisade parenchyma (PP) and lacunous parenchyma (LP) of the 10 best images were determined. The values obtained with the measurements of the 10 repetitions were used for statistical analysis and for calculating the averages.
At 60 days after the first application, photosynthetically active leaflets were collected from the treatments. Evaluation of the preference of TSSM from plants sprayed with different concentrations of SA was carried out in a controlled environment at the Entomology Laboratory of the State University of Midwest. Leaflets from plants kept in a greenhouse were used as described above. Leaf discs (20 mm in diameter) from each treatment (25 mg · L−1, 50 mg · L−1, 75 mg · L−1 and 100 SA mg · L−1) were compared with the control treatment (0 mg SA · L−1). The leaf discs were placed side by side in a Petri dish (60 mm in diameter), connected by a plastic coverslip 2 cm long and 0.08 cm wide. A layer of cotton saturated with water was used on the inner base of the Petri dish to prevent dehydration of the leaflet (Figure 1).
The two foliar discs were connected by a plastic coverslip (18 × 18 mm) in which six TSSM adult females were released using a fine bristle brush. The females were carefully placed in a medium portion of the coverslip to allow access to both the foliar disks. The Petri dishes were then placed in an environmental chamber (25 ± 1ºC; 70 ± 10% RH and photoperiod 12:12 h, light:dark) for 24 h. Later, the females in each disc were counted and recorded. The experimental design was a completely randomized design (CRD) with 10 replicates per treatment.
For the no-choice bioassay, leaf discs (30 mm in diameter) from each treatment were used, as mentioned above. A leaf disc was placed in the centre of each Petri dish, on which one female TSSM was released from the controlled creation (Figure 2). The Petri dishes were placed in an environmental chamber (25 ± 1ºC; 70 ± 10% RH and photoperiod 12:12 h, light:dark). The number of eggs and live adults were counted 24 h after release (Goldstein et al., 1992). The experimental design was a CRD with 10 replicates per treatment.
Normality and homoscedasticity tests were performed on the data obtained. After verifying the assumptions, ANOVA at
In general, all strawberry morpho-anatomical characteristics were affected by the application of SA at
The maximum number of glandular (20.97) and non-glandular (28.41) trichomes per sample (300 μm2) was estimated at 70.00 mg · L−1 and 56.98 mg · L−1 of SA, respectively, with the lowest densities being observed in the control treatment (0 mg · L−1) and the highest concentration (100 mg · L−1) as shown in Figure 5. The maximum thickness of leaflets (180.37 μm) and mesophyll (125.52 μm) was estimated at 63.79 mg · L−1 and 54.03 mg · L−1 of SA, respectively. Also, the highest values for abaxial cell wall (49.57 μm), adaxial cell wall (48.27 μm), palisade parenchyma (81.06 μm) and lacunous parenchyma (56.12 μm) were observed from 52.96 mg · L−1 and 71.16 mg · L−1 of SA.
The observed frequencies in the two-choice assay were significant at
The variable number of live females and laid eggs fit the quadratic adjustment in the regression equations in function of the SA concentrations (Figure 7). A maximum reduction in the number of live females (1.11 per mm2) was found for SA at 54.38 mg · L−1. The smallest number of eggs (7.41 per mm2) was recorded at 54.88 mg · L−1 SA.
The TSSM number of laid eggs and live females was correlated negatively with the morpho-anatomic traits (Figure 8). Also, the number of laid eggs was strongly negatively correlated with palisade parenchyma, and adaxial and abaxial cell wall thickness. A moderate strong correlation between the number of laid eggs and the glandular and non-glandular trichomes was also found. The number of live females was strongly negatively correlated with the palisade parenchyma thickness and the number of glandular and non-glandular trichomes. Moreover, the thickness of the adaxial cell wall, mesophyll and leaflet were moderately correlated with the number of live females.
The first and second principal components (PC1 and PC2) explained 65.8% and 11.1% of the total variation, respectively (Figure 9). From the PCA biplot, it is possible to observe that the vectors of characteristics MT, NGT, PP, LT, LP, AbCW, AdCW and GT were associated with the 50 mL · L−1 concentration, indicating that the highest averages of these characteristics were obtained in this SA concentration. However, the vectors of characteristics NE and NM were associated with the 0 mL · L−1 concentration. In general, the PCA agreed with the results presented by regression and correlation analyses.
This study aimed to investigate the effect of exogenous SA on morpho-anatomical changes in strawberry leaves and induced resistance against
Increasing leaflet thickness, mesophyll, lacunous and palisade parenchyma suggests that SA may act to stimulate cytokinin for gene expression by producing enzymes via chlorophyll biosynthesis, chloroplasts and ribulose 1–5 biphosphate – Rubisco production (Cortleven and Schmulling, 2015; Vishwakarma et al., 2018). This enzyme is associated with CO2 fixation (Calvin cycle) producing glyceraldehyde 3-P (triose-P), pectin and lignin, which are deposited in the cell wall (Herrero et al., 2013; Ye et al., 2018) and possibly associated with plant resistance against biotic stresses (Baxter and Stewart, 2013). Thickness in morphological structures usually make it difficult to feed sucking pests such as mites and aphids. This fact is evidenced by the strong correlations estimated between mite survival and thickness of the parenchyma. It is inferred that the mite showed greater resistance to penetration from the cheliceras to the cleaved tubes. Inhibiting the penetration of the pest mouthpiece into cellular content is an important form of plant defence. Young larvae of
Regarding chickpeas plants, exogenous SA treatment increased the activity of glutamine synthetase, glutamate synthase and hydrogenase glutamate, suggesting stimulation of the nitrogen metabolism as shown previously. Subsequently, higher amino acids and proteins are synthesised to structure the plant morphology and anatomy, besides other functions (Birch, 1984; Conesa et al., 2020).
Increasing the number of glandular and non-glandular trichomes due to exogenous SA treatment agrees with other previous investigations on some plants such as tomato (Chen et al., 2018),
The current study showed that the highest SA concentrations diminished the magnitude of morpho-anatomical changes in strawberry leaflets. Higher SA concentrations in tissues have been suggested as inhibiting gene transcription responsible for syntheses of structural proteins, mediated by jasmonic acid (Caarls et al., 2017) due to the antagonistic action of phytohormones (Ederli et al., 2020). Moreover, higher SA doses induce high levels of oxygen reactive species (ROS) and cause decreasing detoxification capacity, cell death and oxidative stress, which triggers an imbalance in physiological and biochemical plant activities (Otaiza-González et al., 2020).
Similarly, intermediate SA concentrations determined the highest change in TSSM responses in both two-choice and no-choice assays. Reduction in the number of eggs and live females in the no-choice assay reached 75% and 84.6% compared with the control, respectively. Treated strawberry plants with suitable SA concentrations may induce antixenosis and antibiosis on TSSM. Due to treatment with SA, the two-choice assay mites showed oviposition and permanence, in the test with a chance of choice, indicating that it is a resistance mechanism of the type antixenosis or no preference. The reduction in the number of eggs and live adult individuals on the surface of the leaflets with higher concentrations of SA application, in the test with no-choice, indicates that there was an influence of the treatment on the biology of the pest, and therefore, it is a mechanism of antibiosis resistance. Intermediate concentrations of SA were effective in reducing mite survival and oviposition in strawberry cultivars Aromas and Sweet Charlie, as observed in the present study (Favaro et al., 2019).
The influence of the strawberry foliar glandular and non-glandular trichomes on the resistance to TSSM was suggested by other researchers (Figueiredo et al., 2013; Kumari et al., 2018; Resende et al., 2020). Non-glandular trichomes function as a system of mechanical defence, diminishing oviposition, feeding and locomotion (Krimmel, 2014; Riddick and Simons, 2014), whereas glandular trichomes secrete and release defensive secondary metabolites such as terpenes, alkaloids and phenols (Delgoda and Murray, 2017; Tian et al., 2017). A significant and positive correlation between the trichome densities and TSSM infestation in strawberry cultivars was also previously noted (Esteca et al., 2017). The highest trichome density was found on ‘IAC Princesa Isabel,’ ‘IAC T-0104,’ and ‘IAC 12’ cultivars which were more resistant than ‘IAC Guarani,’ ‘Oso Grande,’ and ‘Albion’ (lesser trichome density) (Esteca et al., 2017). Glandular trichomes may also contain defensive substances that constitute a first-line chemical barrier against infesting arthropods. The sesquiterpenes Zingiberene, present in some tomato glandular trichomes, was associated with TSSM nymph mortality and diminishing fecundity (Oliveira et al., 2018; Oliveira et al., 2020). The strong correlation observed between the density of non-glandular trichomes and the oviposition and survival of adults are presumably related to the dysfunction caused by these structures to the movement and permanence of the mite on the surface of the leaflet (Resende et al., 2020).
Meanwhile, the thickness of the structures, as induced by proper SA concentration, may also constitute a barrier to TSSM and consequently reduce feeding, since before reaching the mesophyll, peripheral structures must be pierced and crossed by the stylet (Schmidt, 2014; Seki, 2016). Association between thicker foliar structure in barley (
The correlations indicate that the morphological characteristics directly interfered with the pest behaviour, decreasing oviposition and survival. The increase in the number of trichomes, glandular and non-glandular, correlated significantly and negatively with the oviposition and survival of the mites, as well as the thickness of the mesophilic abaxial surface and thickness of the palisade and lacunous parenchyma. The increase in non-glandular trichomes behaves like a physical barrier, making feeding and displacement on the surface difficult. Glandular trichomes release substances that repel or increase pest mortality (Antonious and Snyder, 2006). The ability to accumulate compounds such as terpenes and tannins (Rattan, 2010) and the production of oxidising enzymes involved in the oxidation and polymerisation of phenolic compounds, act as chemical barriers against the feeding of mites and indirectly affect their oviposition (Steinite and Ievinsh, 2003). To suck the plant's cellular liquid, the mite punctures the epidermis of the abaxial surface of strawberry leaves, causing cell damage (Fadini et al., 2007). The rupture of these cells results in the leakage of cell content, ending with cell death. Dead and injured cells cause physiological disorders, such as increased sweating rates and inhibition of starch synthesis in the chloroplast, resulting in the accumulation of starch precursors, such as D-glucose, in the cytoplasm. Thus, thicker morpho-anatomical structures, such as parenchyma, cell wall and mesophyll, decrease the penetration of the oral apparatus of pests.
Regarding the mode of action, it is well known that SA is one of the modulators of systemic acquired resistance (SAR) on plants by activation of specific pathways that affect the gene expression and lead to the production of substances with a defensive proposal. In general, activation of the resistance modulated by SA on plants is possible since plants may detect abiotic and abiotic stressing factors (Yusuf et al., 2013) that trigger genetic, metabolomic and physiological changes. In the same vein, various secondary metabolite compounds may be produced by activation of the defence signalling pathways such as anthocyanin (Ran et al., 2013), rosmarinic acid (Sahu, 2013), terpenoids (Saiman et al., 2015) and flavonoids (Lee, 2010). A plethora of compounds are involved in TSSM plant resistance, as reviewed by Santamaria et al. (2020). SAR on plants mediated by exogenous SA may also cause increased production and release volatile repellents such as MeSa and δ-limonene, which repel
Using SA for TSSM management in strawberry crops may be tested in future studies due to the negative effects of treating plants with SA on TSSM preference and biology (Figures 4 and 6). SA could be applied when the environmental conditions – dry and warm weather – are favourable to TSSM development and could eventually be used together with other measures/methods to minimise TSSM infestations such as biological control (Rowen et al., 2017) or intercropping systems (Hata et al., 2019). Under such conditions, strawberry flowering is also negatively affected. SA had been suggested as an activator of heat thermo-tolerance mechanisms (Hameed and Ali, 2016); further investigations could address these features.
Intermediate concentrations of SA promoted morpho-anatomical changes in the strawberry leaf, increasing the thickness of the leaf structures and the number of glandular and non-glandular trichomes. There was a significant correlation between the SA doses and the resistance to the two-spotted spider mites, probably mediated by the morpho-anatomical changes in the leaves. The application of high doses of SA promotes an antagonistic effect, making the plant more susceptible.