In terrestrial ecosystems, plants are the link between below- and above-ground communities, which may in consequence either indirectly, by altering plant traits, or directly, by moving up or down in the plant, interact with each other. During the last two decades, research on below-aboveground interactions has immensely intensified and many studies have focused on effects that cascade up and down between the communities of both subsystems (Van der Putten et al., 2001; Pineda et al., 2010; Hoffmann et al., 2011a,b; Friman et al., 2021b). Plant-mediated below-aboveground interactions involving micro-organisms of the rhizosphere may occur between plant-associated and/or free-living microorganisms (Bezemer and van Dam, 2005; Coskun et al., 2017; Dellagi et al., 2020), between micro-organisms and herbivores (Hoffmann et al., 2009; Patiño-Ruiz and Schausberger, 2014; Disi et al., 2019; Friman et al., 2021a) and, in turn, between micro-organisms and the herbivores’ natural enemies (Hoffmann et al., 2011a,b; Pineda et al., 2013; Gadhave et al., 2016). The rhizosphere microbial community may consist of a huge diversity of pathogenic, neutral and/or mutualistic micro-organisms. Among the usually mutualistic symbionts, it is especially mycorrhizal fungi, rhizobia and plant growth-promoting rhizobacteria (PGPR) that may influence aboveground plant growth and defense against pathogens and herbivores (Hoffmann et al., 2009, 2011a,b; Hoffmann and Schausberger, 2012; Khaitov et al., 2015; Pangesti et al., 2015; Nunes and Kotanen, 2017; Friman et al., 2021b). Here, we studied plant-mediated interactions between free-living soil bacteria that are commonly considered PGPR and aboveground herbivorous spider mites on strawberry plants.

Free-living soil bacteria such as Azotobacter spp., Pseudomonas spp., Azospirillum spp., and Bacillus spp. are well adapted to establish and thrive in competitive rhizosphere environments. Among other activities, these bacteria are well-known for using different kinds of inorganic nitrogen (N) from the soil and/or fixing atmospheric nitrogen (N₂) (Steenhoudt and Vanderleyden, 2000; Dellagi et al., 2020), and thereby provide for nitrogen availability for uptake by the plants. Hence, free-living rhizobacteria can support plant growth and development via optimizing uptake of nitrogen and other nutrients such as phosphorus, iron and other micronutrients from the soil (Kempel et al., 2009; Gamalero and Glick, 2011). Due to the globally increasing demand for high agricultural productivity and the awareness of the disadvantages of using synthetic chemical pesticides and inorganic chemical fertilizers, ever growing efforts have been dedicated to sustainable agriculture (Gamalero and Glick, 2011). Application of biofertilizers such as PGPR to supply all or parts of nutritional requirements of crops is becoming more and more popular (Erturk and Çakmakçı, 2012; Conti et al., 2014; Andrade et al., 2019).

In addition to, and/or as a consequence of, promoting nutrient uptake by plants, PGPR may influence plant defense against below- and above-ground pathogens and herbivores. The phenomenon of belowground micro-organisms priming plants for defense against later occurring threats, or inducing a defense response against biotic stressors of aboveground plant parts, has been dubbed induced systemic resistance (ISR) (Dam, 2009; Glick, 2013; Pieterse et al., 2014). ISR is elicited by beneficial microbes in the rhizosphere and within-plants regulated by a network of endogenous signaling pathways, in which phytohormones play a major role (Pineda et al., 2010; Pieterse et al., 2014). Several studies have addressed indirect plant-mediated interactions between rhizobacteria and above-ground herbivores’ performance (Zehnder et al., 1997; Khan et al., 2018). These interactions are highly system- and context-specific and may range from positive (Khaitov et al., 2015) to neutral (Valenzuela-Soto et al., 2009) to negative (Zamioudis and Pieterse, 2012) effects on the herbivores, among others depending on the species of bacteria, plant and herbivore, the defensive array of the plant and the abiotic environmental conditions (Shah et al., 2021). Free-living rhizobacteria may increase the abundance of herbivores by enhancing the plant’s nutritional state but may simultaneously enhance growth and biomass of vegetative shoot and root tissue (Khaitov et al., 2015). Thus, rhizobacteria may affect plant tolerance to herbivore infestation by enhanced regrowth following herbivore injury (Disi et al., 2019). Free-living rhizobacteria can also trigger or enhance biosynthesis of defense-related chemical compounds in plants and, by this way, decrease life history performance and population growth of herbivores attacking aboveground plant tissue (Fahimi et al., 2014; Khoshfarman-Borji et al., 2020; Pourya et al., 2020).

Strawberry, Fragaria × ananassa Duchesne (Rosaceae), is one of the most frequently cultivated fruit crops around the world (Todeschini et al., 2018). Several studies documented that treatment of strawberry plants with PGPR may improve plant growth and reproduction (Martín et al., 2007; Pırlak and Köse, 2009; Roussos et al., 2012). Additionally, PGPR application may increase the concentration of some polyphenolic compounds in strawberry plants (Lingua et al., 2013; Todeschini et al., 2018), which may protect the plants against biotic stressors. However, the effects of PGPR on aboveground herbivores of strawberry have not yet been investigated. Two-spotted spider mites Tetranychus urticae Koch (Acari: Tetranychidae) are ubiquitous herbivores and highly destructive pests of a wide variety of agricultural and horticultural crops and may cause significant yield loss in field- and greenhouse-grown strawberry plants (Wermelinger et al., 1985; Migeon et al., 2010; Alizade et al., 2016). Severe spider mite infestations may strongly reduce strawberry fruit quality and quantity (Attia et al., 2013). While the effects of rhizosphere fungi on aboveground-feeding spider mite performance have been investigated in various plants such as bean (Hoffmann et al., 2009, 2011a; Hoffmann and Schausberger, 2012; Patiño-Ruiz and Schausberger, 2014), tomato (Pappas et al., 2018), and pepper (Pappas et al., 2021), it is unknown whether the presence of PGPR in the soil influences the defense system of strawberry plants against spider mites. This is an important question because spider mites usually thrive and are favored by high N-contents of plants (Wermelinger et al., 1985, 1991) such as presumably provided by PGPR (Khaitov et al., 2015 for common bean Phaseolus vulgaris L.).

Accordingly, we investigated whether treatment of strawberry plants with the PGPR Azotobacter chroococcum Beijerinck, Azospirillum brasilense Tarrand, Krieg & Döbereiner and Pseudomonas brassicacearum Achouak et al. positively affects the nutritional state of strawberry plants, which could either make them more prone to attack by two-spotted spider mites and enhance their performance and/or, additionally or alternatively, induce and/or strengthen the strawberry defense system against herbivory and thereby decrease the performance of spider mites. In detail, we scrutinized the effects of PGPR treatments on strawberry plant growth and physiology and the life history and population dynamics of T. urticae feeding on strawberry leaves and plants growing in substrates treated with PGPR, as compared to plants treated with chemical fertilizer and untreated plants (control).

Below-ground beneficial micro-organisms may affect the performance of above-ground herbivores through plant-mediated mechanisms that may involve changes in plant physiology including enhancement of the plant’s nutritional state and/or induction of systemic resistance (ISR). The latter often results involves the production of secondary plant metabolites (Dean et al., 2009; Hoffmann et al., 2009; Kempel et al., 2009; Khaitov et al., 2015; Ruiu, 2020; Friman et al., 2021a). There exists ample evidence documenting that below-ground interactions with beneficial micro-organisms enhance nutrition and growth of strawberry plants (e.g., Pırlak and Köse, 2009; Todeschini et al., 2018; Andrade et al., 2019); however, the effects of these belowground interactions on life history performance and population dynamics of the cosmopolitan and most serious herbivorous pest of aboveground strawberry plant tissue, T. urticae, have not yet been studied. Here, we document adverse plant-mediated effects of three species of free-living rhizobacteria, Azotobacter chroococcum (Ac), Azospirillum brasilense (Ab) and Pseudomonas brassicacearum (Pb), on life history and population dynamics of T. urticae feeding on strawberry leaves and plants, and positive effects of these rhizobacteria on flowering and physiology of whole strawberry plants. Spider mites feeding on rhizobacteria-treated strawberry plants had longer developmental times and lower fecundity in comparison to those feeding on untreated and chemically fertilized plants. Overall, the intrinsic rate of increase (r_m) of T. urticae was the lowest on Ac- and Ab-treated strawberry leaves and the highest on chemically fertilized strawberry leaves. Young rhizobacteria-treated clean plants produced more flowers than untreated and chemically fertilized plants but had similar numbers of leaves; stomatal conductivity tended to be higher in bacteria (especially Pb)-treated plants than in untreated and chemically fertilized plants whereas chlorophyll content was similar in Ac-treated and untreated plants and higher in chemically fertilized, and Ab- and Pb-treated plants.

Population dynamics of spider mites growing on PGPR-treated, chemically fertilized and untreated whole strawberry plants was in accordance with the adverse PGPR effects on spider mite life history. The mean abundance was lower in spider mite populations feeding on PGPR-treated strawberries than in those feeding on chemically fertilized and untreated plants. Time-dependent population development fluctuated similarly in all treatments except for the chemical fertilizer treatment. Mite abundance dropped to relatively low levels in the chemical fertilizer treatment in week 4 but grew exponentially from week 5 onward (corresponding to the 2nd and 3rd spider mite generation after transfer to the treated plants), which was expected considering the high intrinsic rate of increase. The drop in abundance in week 4 was probably caused by the higher nutrient content of leaf tissue of chemically fertilized plants making the mites staying longer on older leaves, and dispersing later to the newest leaf, than in the other treatments (weekly sampling concerned always the youngest fully developed leaf).

Beneficial below-ground microorganisms may influence the performance of herbivores via two major functional pathways, that is, enhancement of nutrition promoting plant growth and development, which in turn may favor the herbivores, or induced systemic resistance (ISR), which may disfavor the herbivores, or the combination thereof (Kempel et al., 2009; Pineda et al., 2010; Gamalero and Glick, 2011; Friman et al., 2021b). Many studies of above-belowground interactions showed that beneficial soil microorganisms such as free-living rhizobacteria can induce systemic resistance in above-ground plant parts and enhance the level of protection against herbivorous attackers (Zehnder et al., 1997; Koricheva et al., 2009; Khoshfarman-Borji et al., 2020; Ruiu, 2020; Friman et al., 2021a). Regarding interaction between the three species of rhizobacteria used in our study and aboveground herbivores, nothing has been known for P. brassicacearum (Pb). A. brasilense (Ab) had no effects on Hessian flies, Mayetiola destructor, in wheat (Prischmann-Voldseth et al., 2020) and soybean aphids Aphis glycines (Brunner et al., 2015) and positive effects on armyworm Mythimna separata in maize (Li et al., 2018). A. chroococcum (Ac) exerted positive effects on spider mites T. urticae in common bean (Khaitov et al., 2015) and both negative and positive effects (context- and plant genotype-dependent) on armyworm M. separata in maize (Li et al., 2018; Song et al., 2020).

In general, ISR may involve changes in the secondary metabolite profile such as alkaloids, phenols, terpenes, and flavonoids, some of which may inhibit feeding or digestion or have other toxic activity against herbivores (Pineda et al., 2010; Gadhave and Gange, 2013; Disi et al., 2019; Ruiu, 2020). For instance, reduced larval feeding and development of the beet armyworm Spodoptera exigua (Hübner) on Bacillus spp.-treated cotton plants was mediated by increased biosynthesis of gossypol, a phenolic aldehyde with insecticidal property, as compared to untreated plants (Zebelo et al., 2016). Our study revealed that both male and female spider mites, and in each life stage, needed longer time to develop on PGPR-treated strawberry plants and had lower immature survival rates than those fed on chemically fertilized and untreated plants. Reduced age-specific fecundity, longer developmental time and lower age-specific survival rates (as estimators of increased intrinsic rate of increase; Vehrsi et al., 1992) of the mites feeding on rhizobacteria-treated plants reduced their intrinsic rate of increase as compared to mites feeding on chemically fertilized and control plants. Collectively, these results suggest that rhizobacteria inoculation induces phytohormonal signaling (jasmonic acid – JA – and ethylene) that regulates the concentration of secondary plant metabolites (Stout et al., 2002; Pineda et al., 2010, 2013; Pieterse et al., 2014). For example, Steinite and Levinsh (2002, 2003) showed that induced resistance in strawberry, indicated by increased polyphenol oxidase, which is an important JA-inducible marker, negatively affected reproduction of T. urticae. Mouden et al. (2021) reported that induction of the JA pathway enhanced strawberry plant defense against T. urticae via increased production of phenolic acids, flavonoid compounds and anthocyanins. Similarly, Luczynski et al. (1990) observed that the development of T. urticae correlated negatively with foliar concentrations of phenolics. Therefore, we assume that in our study system increased biosynthesis of phenolic compounds and other secondary metabolites in rhizobacteria-treated strawberry plants acted as feeding deterrents, digestion inhibitors and/or toxins for the spider mites. Population dynamics of T. cinnabarinus Boisduval was inhibited in response to increased tannin levels (a polyphenol compound) in cotton (Ma et al., 2021). Similar to our study, Alizade et al. (2016) showed enhanced life history performance and population growth of T. urticae on N-fertilized strawberry plants. Increasing N content of the host plant tissue via chemical N fertilization improved the availability of dietary proteins, which typically benefits herbivores such as two-spotted spider mites (Alizade et al., 2016; Hosseini et al., 2018). Beneficial soil bacteria commonly also improve plant nutrition via increasing the uptake and concentration of a variety of nutrients, including nitrogen, and might also enhance the production of phytohormones that promote growth indicators like the rate of photosynthesis activity (Zhang et al., 2008; Pineda et al., 2010). In our study, application of rhizobacteria increased photosynthesis and growth compared to control plants, resulting in leaf chlorophyll content of Ab- and Pb-treated strawberry plants being similar to that of chemically fertilized plants and PGPR treatments having higher stomatal conductivity than chemically fertilized plants; while vegetative growth, indicated by the number of leaves, did not differ among treatments, reproductive growth, measured as the number of flowers, was significantly enhanced by rhizobacteria application, relative to chemically fertilized and untreated control plants.

Due to possible trade-offs between investment in growth and defense, the positive effects of mutualistic belowground microorganisms–plant interactions on plant growth do not necessarily and not always result in a net fitness benefit for the plant if the fitness of plant-associated herbivores is also enhanced and third trophic level natural enemies are left out (Gehring and Whitham, 2003). We did not assess the net effect of the rhizobacteria on plant growth at simultaneous defense against herbivores, because we just measured the plant characteristics of clean plants; however, our study documents that rhizobacteria can principally enhance both reproductive growth and defense of strawberry plants. Accordingly, we assume that rhizobacteria application may help attacked strawberry plants to limit the damage caused by spider mites, via inducing or increasing the production of secondary metabolites interfering with mite proliferation (Alizade et al., 2016), and to alleviate the deleterious effects of mite damage, via enhancing plant tolerance through vegetative or reproductive regeneration of herbivore-injured plant tissue, as a result of an improved nutritional state (McNaughton, 1983; Disi et al., 2019).

Overall, our results stress the importance of soil microbe-plant interactions on life history and population dynamics of aboveground herbivores. Rhizobacteria inoculation was more favorable than chemical fertilization in terms of decreasing the life history performance of T. urticae and limiting its population growth and improving reproductive growth of strawberry plants. Our study suggests that in crops with well-developed direct anti-herbivore defense systems, such as strawberry, PGPR can efficiently contribute to dampen herbivore infestation and provide for an improved nutritional state of the host plant. Future investigations should explore the consequences of PGPR application on aboveground tri-trophic interactions.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AH and MH conceived the study idea and designed the experiments. AH conducted the experiments, analyzed the data, and wrote the first draft of the manuscript. MH contributed to methods development and managed the project locally. PS gave advice on experimental design and contributed to results analysis and presentation. MH and PS contributed to writing and wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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