Relationship between salicylic acid and resistance to mite in strawberry

The total area for worldwide strawberry (Fragaria × ananassa Duch.) harvests is estimated to be about 395,844 ha, which amounts to circa 9,125,913 tons (FAOSTAT, 2019). The striped mite (TSSM) Tetranychus urticae Koch (Acari: Tetranychidae) is a polyphagous pest found on cultivated and uncultivated plants (Osman et al., 2019). So far, TSSM is found to be the most deleterious pest that attacks strawberries in a protected environment all over the world (Esteca et al., 2017; Ong et al., 2018). TSSM causes qualitative and quantitative losses in strawberry production; especially during periods of low rainfall (Dias et al., 2012; Rezaie et al., 2013), TSSM has increased reproductive potential and a decreased life cycle, which leads to an increase in the infesting population, and consequently, extensive damage being caused to the commercial crop. Due to increase in the pest population, there is increase in the application of pesticides, which not only promotes the development of pest resistance to phytosanitary treatments, but also increases the damage to the environment and to the health of farmers and consumers (El-Wakeil, 2013; Wang et al., 2018). Moreover, it is well known that pesticides/acaricides have several limitations, generating a biological imbalance and consequently, increasing the costs of production (Koch et al., 2017; Silva et al., 2019). Thus, there is a strong interest in the development of alternative methods of control, which are more sustainable (Lachhab et al., 2015; Youssef et al., 2019).

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 T. urticae.

This study aimed to investigate the effect of exogenous SA on morpho-anatomical changes in strawberry leaves and induced resistance against T. urticae. The findings reported in this article evidence that exogenous application of SA markedly affects the morpho-anatomic traits in a dose-dependent manner in strawberry leaflets. The morpho-anatomical changes can be explained by the direct SA effects on cell division and expansion (Kumari et al., 2018; Lu et al., 2016). Intermediated concentrations were the most suited to promote these changes. The magnitude of the differences was 42.9%, 60.1%, 41.19%, 43.20%, 45.10%, 48.00%, 66.30% and 33.3% times higher than the control for thickness of the leaflet, mesophyll, lacunous and palisade parenchyma, cell wall (abaxial and adaxial) and number of glandular and non-glandular trichomes, respectively. Previously, SA was reported to regulate lignin deposition (Gallego-Giraldo et al., 2011; Tenhaken, 2015; Zhu, 2016), triggering hardness and thickness of the secondary cell wall.

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 Aphis fabae (Scopoli), Acyrthosiphon pisum (Harris) and Megoura viciae (Buckton) were unable to penetrate the cuticle of wild species of Vicia sp, thus not advancing to the next instar (Birch, 1984).

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), Arabidopsis thaliana (Traw and Bergelson, 2013) and Artemisia annua (Kumari et al., 2018), which may confer resistance to pests (Aziz and Kapoor, 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 (Hordeum vulgare L.) and diminishing TSSM feeding was observed previously due to disturbing stylet penetration into the leaf mesophyll (Gómes Sánches et al., 2018; Santamaria et al., 2020). A negative correlation between TSSM development and foliar thickness was also reported in melon plants (Cucumis melo L.) (Xu et al., 2019). Also, foliar thickness, mostly when associated with some biochemistry traits, was also associated with a reduction of Bemisia tabaci whitefly on egg plants, since negative correlations were observed for oviposition and the number of nymphs and adults (Khan et al., 2018).

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 Bemisia tabaci biotype B on tomato plants (Shi et al., 2016) as a direct defense, attract natural enemies such as pests or even communicate with neighbourhood plants as an indirect defence (Rowen et al., 2017). The magnitude of the morpho-anatomical leaf changes on SA treated plants (Figures 3 and 4) suggests that, together with chemical changes, structural alterations may play a tiny role in SAR mediated by SA and deserve to be studied more deeply.

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.