Natural biocrust communities of arid soils are constituted by hundreds of species showing adaptation traits to local stresses (Belnap, 2003; Evans and Johansen, 1999). Selected isolates can be used as inocula, exploiting their adaptation to the environmental conditions of the reclamation site. When inoculated, isolates may exhibit both bioconditioning and biofertilizing capabilities or, more commonly, either one or the other (Maqubela et al., 2010). However, when isolates are reintroduced in their native soil, they may display a different capability to establish and produce significant inoculation effects (Rossi et al., 2017). To survive under high-abiotic-stress conditions after being dispersed in the soil, the inoculant must possess suitable physiological stress-tolerance traits and, in particular, the capability of constituting stable and erosion-resistant bio-aggregates. It is crucial to perform screenings at laboratory scale and under natural outdoor conditions to test the effects of drought, UV irradiation, and high saline concentration, which are typical of high-abiotic-stress environments. The tolerance to natural conditions can be improved by “hardening off”, namely the subjecting of cyanobacterial candidates to preliminary preconditioning treatments consisting of several dry–wet cycles and progressive increase in illumination (Giraldo-Silva et al., 2019) before inoculating them in the field. Isolates may show either good, fair, or limited capability to form physically stable cyanobacterial biocrusts in short times. Indeed, a positive assessment of stress tolerance traits does not necessarily guarantee a biocrust-forming capability, which has to be separately assessed. For example, Nostoc spp. and Scytonema javanicum synthesize UV-screening pigments (Garcia-Pichel and Castenholz, 1991, 1993), are capable to easily recover from UV-related damages (Chen et al., 2013), and have drought and salt stress defensive mechanisms (Tamaru et al., 2005; Tang et al., 2007). However, these strains were far worse sand stabilizers of arid sandy soils than Microcoleus vaginatus, which does not possess any of these traits (Hu et al., 2002). The quick soil-stabilizing capability has relevance depending on the strength of the erosive forces in the site to reclaim (Park et al., 2015). In environments characterized by high wind speed and unconsolidated soil, biocrusts with a significant wind threshold friction velocity are able to endure and not be worn away. Some natural biocrust-dwelling non-heterocystous genera such as Microcoleus, Leptolyngbya, and Schizothrix are proficient sand stabilizers and have been proven to induce biocrusts with significant aggregate stability and tensile strength (Hu et al., 2002, 2012; Mugnai et al., 2018a, b). In particular, strains with the capability to glide in the substrate (e.g., M. vaginatus, Leptolyngbya ohadii) seem to have an advantage and more apt to determine the formation of stable and thick biocrusts (Mugnai et al., 2018b). The capability to migrate in the soil allows for adaptations to drought and intense light. For example, when topsoil is moistened, M. vaginatus glides to the soil surface, leaving its hygroscopic sheath behind, and then retracts in it when the soil dries and when the light is too strong (avoidance). The strain can also accommodate at the soil depths where light intensity is optimal (Hu et al., 2003). Following these movements, sheath material is distributed in the soil, cementing soil particles (Belnap, 2003). On the other hand, non-gliding genera such as Tolypothrix spp., Nostoc spp., and Scytonema spp. are localized at the topsoil and synthesize sunscreen molecules to protect from UV radiation (Román et al., 2018). While being less valid sand stabilizers than M. vaginatus or Phormidium tenue (Hu et al., 2002), diazothrophic non-motile strains proved to be valid inoculants on substrates with a higher content in silt and clay where they exert a significant fertilizing effect (Chamizo et al., 2018; Román et al., 2018; Roncero-Ramos et al., 2019a, b). In order to attain both biostabilizing and biofertilizing effects, strains with a marked biofertilizing capability such as S. javanicum can be co-inoculated with strong biostabilizing strains such as M. vaginatus. The co-inoculation of M. vaginatus and S. javanicum (10:1) was successful in some large-scale studies conducted in Hopq Desert, China (Chen et al., 2006; Lan et al., 2014). S. javanicum greatly contributes to dinitrogen-fixing activity and the maintenance of soil productivity (Deb et al., 2015).

Cyanobacterization studies generally report non-uniform protocols for inoculum preparation and dispersion (Nelson et al., 2020). Cyanobacterial inocula have consisted of wet or dry forms, with or without specific formulations (i.e., the addition of specific carriers and amendments). The biomass has been inoculated alone (no formulation) or mixed with sand, lime, gravel, or superabsorbent polymers or encapsulated in alginate or extruded soil pellets to enhance soil aggregation and prolong inoculum shelf life (Buttars et al., 1998; Park et al., 2015; Román et al., 2020a; Singh, 1961; Hamdi, 1982). Overall, there is a lack of exhaustive investigations examining how differences in the methodological approach can affect inoculation cyanobacterial survival and biocrust formation. Cyanobacteria can respond differently to inoculum preparation treatments, such as the desiccation process associated with pelletization (Román et al., 2020a). As an example, in microcosms, we observed how M. vaginatus develops biocrusts if inoculated as liquid biomass, while the same powdered inoculum did not produce significant aggregation even though followed by watering (our unpublished data). Although further study is needed to provide valid explanations, the reason could be related to the different physiological responses to the biomass treatment to remove water. It is possible that a forced drying process does not allow the drought-response systems – presumably EPS synthesis, compatible solute accumulation, down-regulation of photosynthesis, N fixation, RNA polymerase, and other processes (Singh, 2018) – to be fully activated, hence affecting cell viability after rehydration once inoculated. The level of homogeneity of biomass dispersion is another factor that can affect the development of the biocrusts. In microcosm it has been demonstrated that L. ohadii produced biocrusts with the highest consistency and stability when the inoculum was distributed as homogenously as possible over an entire available surface of the microcosm (Mugnai et al., 2020). A further parameter of importance is the optimal inoculum concentration, which has to be experimentally determined according to the strain physiology but also in relation to substrate characteristics. We have observed how the larger the grain size of the substrate, the higher the amount of inoculum per square centimeter needed to obtain cyanobacterial biocrusts with significant hydraulic and tensile strength (Mugnai et al., 2020). Figure 2 shows biocrusts induced in sand microcosm by inoculating Schizothrix delicatissima AMPL0116, with a typical morphology deriving from a topsoil-dwelling inoculant, characterized by cracks and curls on the surface (Fig. 2).

The inoculation at a large field scale requires elaboration of cost-effective methodologies to cultivate large volumes of culture. In most case studies, biomass was produced using conventional culturing systems characterized by stepwise culture vessel volume increments (Guedes et al., 2013). However, classical methods are inherently expensive owing to the need of producing large volumes of culture medium and may not fit bundle-forming cyanobacteria that form clumps and have their growth hindered. In order to reduce implementation costs, the elaboration of cheaper culture media and suitable bioreactor designs should be pursued. For example, Roncero-Ramos et al. (2019a, b) demonstrated the possibility of growing Nostoc commune in a medium made with fertilizers detecting no negative impacts on inoculation effects. The use of circular open mixing raceway ponds (Liffman and Paterson, 2013), allowing the exploitation of direct sunlight, has been used to grow cyanobacterial biomass for large-scale cyanobacterization in Inner Mongolia, China (Rossi et al., 2017). The inoculum, initially placed into smaller (1 m × 4 m) raceway ponds, is then transferred to larger ponds (6 m × 40 m). The ponds are filled with nitrogen-free culture medium and stirred with one or more electrically driven paddle wheels (Zhou et al., 2020). This system easily applies to mixed cultures, being characterized by non-sterile conditions. On the other hand, it imposes a control for contamination. Lan et al. (2015) demonstrated that controlling temperatures and illumination regimes can limit contamination and be beneficial to cyanobacterial growth. It was found that temperatures below 30 ∘C and light intensity between 600 and 700 µE m-2 s-1 (Liu, 2013) were the best conditions to restrain contamination by microalgae or protozoans.

Giraldo-Silva et al. (2019) solved the problem of bundle formations and consequent growth inhibition of cyanobacteria by successfully inoculating Microcoleus sp. on sterile cellulose tissue floating in growth medium in large petri dishes. The dried cellulose tissue was later shredded to harvest the biomass. This inoculum preparation and production was further improved by the application of a flexible fog-based irrigation system (Nelson et al., 2020).

Another essential step to optimize is the elaboration of a valid inoculum dispersion methodology. Ground dispersal approach using tank truck-mounted sprayers has been used to inoculate a mixed culture of M. vaginatus and S. javanicum on sand dunes of an experimental area in the Hopq Desert, China (Colica et al., 2014). However, this approach hampers a uniform distribution of the biomass owing to landscape natural unevenness and is invasive for the existing flora and microflora owing to the necessity to move dispersal equipment. The possibility of seeding from aircraft dispersal has been hypothesized (Sears and Prithiviraj, 2012), and one related patent already exists (Sears, 2012).

Since natural outdoor conditions can be recreated in the lab only to some extent, further multifactorial experiments are needed to define the optimal culture conditions for obtaining the maximum moss yields under natural conditions. Mosses are very sensitive to disturbance, which can cause change in their coverage, composition, and function (Bu et al., 2015). Therefore, moss inoculation can be challenging in highly disturbed areas where crusts are at early-stage LODs, while representing a valid approach to restore biocrusts at more mature LODs. A lot of abiotic factors can impinge on moss establishment and growth, including the annual rainfall amount, soil pH, soil calcium carbonate content, plant cover, texture, soil organic carbon, and soil texture (Zhao et al., 2009). These factors affect moss establishment according to moss ecotypes and their phenotypic adaptation to the abiotic stresses of their native environment. In general, it has been observed that the scarcity of nutrients and temperature are other significant limiting factors to moss growth. The high sensitivity of mosses to environmental conditions accounts for the limited amount of moss cultivations successfully transferred to the field in China (Zhou et al., 2020). The wide array of parameters affecting moss growth implies the necessity to elaborate species-specific “recipes” for cultivation, considering the single and most significant constraints. The modulation of resource availability and hydration have been investigated in a number of studies. Bu et al. (2018) tested the addition of Hoagland's solution (whose complete formula can be found in Bever et al., 2012) as resource compensation (2.1 L m-2) and found significant positive improvements in moss density in a field plot experiment. This confirms that macronutrients such as K, P, and Ca, also contained in Hoagland's solution, benefit moss growth (Bowker et al., 2005). The study of the best nutrient formulation is fundamental to determine the most beneficial effects on a specific moss species. An example study is represented by that of Yang et al. (2019). This research group tested the effects of different types of nutrient solutions, namely Knop's solution, Murashige–Skoog (MS), Benecke, Part and Hoagland's solution, glucose and sucrose, and the plant growth regulators thidiazuron, 6-benzylaminopurine, and naphthaleneacetic acid on biocrusts dominated by D. vinealis, native to the Chinese Loess plateau. They observed how Knop's solution, MS, Benecke, and Part and Hoagland's nutrient solutions improved the coverage and plant density of moss crusts, although to different degrees, with Hoagland's nutrient solution being the most effective nutrient amendment. At the same time, they observed a concentration-related inhibitory effect of the thidiazuron and of glucose and sucrose; the addition of 6-benzylaminopurine was ineffective as was the naphthalacetic acid. Finally, they came down to an optimal nutrient medium constituted by Hoagland's solution, low glucose and sucrose concentration (lower than 10 g L-1), and small concentrations of plant regulators (i.e., smaller than 1 mg L-1). Antoninka et al. (2016) also found that fertilization with Knop's solution (Ca[NO3]2, KNO3, MgSO4, KH2PO4, ZnSO4) every 2 weeks was beneficial to the growth of S. caninervis and S. ruralis in North American drylands. Similar beneficial effects of the use of Knop's solution were obtained with Bryum pallescens, B. recurvulum, B. argenteum, and B. unguiculata in China (Zhou et al., 2020).

The optimal growth temperature for moss growth ranges quite widely between 5 and 25 ∘C according to the species and the biome of origin (Duckett et al., 2004). The optimal growth temperature may depend on the climate of origin. The optimal temperature for tropical taxa ranges between 20 and 25 ∘C, while it may range between 10 and 15 or even 5 and 10 ∘C for species from cooler climates; temperatures above 30 ∘C even for short periods can be fatal or at best highly damaging to most taxa (Duckett et al., 2004). B. argenteum-dominated biocrusts collected from Mu Us Sandland, China, were grown at the optimal temperature of 15 ∘C (Bu et al., 2015). Incubation temperature can be tricky, as temperatures slightly outside the optimal value might determine the inhibition of growth. For example, the growth of Barbula vinealis was inhibited above 17 ∘C. However, different growth temperatures may differentially affect the net photosynthetic capacity and temperature relations. For instance, one study supported the inverse relationship between photosynthetic rates and latitude for Polytrichum commune collected in sites near Utqiaġvik and Fairbanks, Alaska; Schefferville and Mont Saint-Hilaire, Canada; and Gainesville, Florida (Sveinbjörnsson and Oechel, 1983). Moreover, it was observed how B. argenteum var. muticum Brid., part of continental Antarctica plant life, could withstand full sunlight without being affected by UVA or UVB (Schroeter et al., 2012), apparently not by shoot acclimation but by the production of new shoots and a constant capability of maintaining photosystem health. This contrasts with more temperate climates, where high light is typically avoided by desiccation.

Indeed, illumination is another paramount factor that has to be controlled. In laboratory settings irradiance is often suboptimal compared to the field, where it reaches values of 500–700 W m-2 in full sun, except for mosses growing in the shade (Duckett et al., 2004). High light and continuous illumination are therefore advised. This has been corroborated by Bu et al. (2015), who demonstrated that low light intensity determined low productivity of EPS and low chlorophyll a contents of cultivated moss biocrusts, while 12 000 lx illumination determined the highest peak of these values. However, this cannot be considered a general rule as, in nature, shaded locations generally determine higher moss yields compared to those that are exposed (Bu et al., 2018; Lan et al., 2014), and in at least one case, like those of S. caninervis, it was demonstrated that light intensity had no impact on the growth (Xu et al., 2008). Due to the biological and physiological differences among bryophyte species and to the adaptation of bryophyte species to their native habitat, the light intensity has to be optimized in each case study.

As previously stated, hydration is a key factor for moss establishment. The optimal amount of water to provide to the inoculum before and after dispersion has to be clearly determined. To avoid vitality loss, moss fragments must receive enough water to photosynthesize and fix enough carbon to balance carbon lost with respiration (Stark et al., 2011). Consequently, hydration must assure an optimal photosynthetic activity. To assure this, as already reported in this review, for example, Condon and Pyke hydrated fragments of B. argenteum and S. caninervis for 20 min prior to fragment dispersion in the field, disregarding the continuous water provision which was guaranteed in the field (Condon and Pyke, 2016). Mosses' tolerance to desiccation can depend on inherent species-specific capabilities or on phenotypic plasticity, which is also responsible for thermal tolerance observed between different ecotypes (He et al., 2016). The factors impinging on desiccation tolerance are the rate of desiccation, the equilibrating water content, and the duration of desiccation, in which moss tissues remain dehydrated. Studies on moss desiccation capability focus both on induced desiccation tolerance, which is characterized by a slow desiccation rate that prepares the tissues for the next drying event, and on the hardening process, which prepares the moss structure to sustain a dehydration event at any speed (Brinda et al., 2016). Hardening has been demonstrated for bryophyte gametophytes with three possible methods: a slow dehydration followed by a rapid dehydration, the treatment with abscisic acid followed by a rapid dehydration, and a partial dehydration followed by a rapid dehydration (Brinda et al., 2016).

Studies such as that of Greenwood et al. (2019) are essential to study the physiological response to desiccation between different ecotypes of the same species. The authors observed a different inherent desiccation tolerance between different B. argenteum ecotypes and a different phenotypic plasticity, especially in the protonemal and juvenile shoot phases. These types of studies pave the way for possibly predicting reaction to climate change of moss inoculants and to set up valid hardening protocols. The large distribution of a species like B. argenteum also depends on genetic variability, which seems constitutive of such species (Pisa et al., 2013).

Large-scale inoculation treatment with cyanobacteria is still quite limited, although some published results showed premises. Cyanobacteria represent the only inoculants that have allowed rehabilitation in 200 ha areas (Zhou et al., 2020). Noticeably, the experiments carried out by the Institute of Hydrobiology (IHB), Chinese Academy of Sciences, Wuhan, China, were carried out in five different deserts, i.e., Qubqi, Ulan Buh, Mu Us deserts, Horqin Sand Land, and the Hulun Buir Sand Land, covering an area of 40 km2 (Fattahi et al., 2020). When inoculating only M. vaginatus, cyanobacterial crusts were visible after 20 d from the inoculation (Chen et al., 2006). The mixed inoculum of M. vaginatus–S. javanicum (10:1) on sand dunes produced cyanobacterial crusts visible in only 1 week (Lan et al., 2014); a well-developed cyanobacterial crust had formed a few years after the inoculation with the mixed cyanobacterial suspension (Colica et al., 2014; Wu et al., 2013). Induced biocrusts in the Qubqi desert were visible well after 13 years following the inoculation (Fig. 3). Moreover, Wu et al. (2013) observed a darkening process on the soil surface over 7 years caused by increased pigment contents, mainly chlorophyll a and scytonemin. Follow-up studies after large-scale inoculation with cyanobacteria evidenced how the induced biocrust system is characterized by the presence of biocrusts at later LODs (moss/lichen crusts) surrounded by early LOD biocrusts (cyanobacterial biocrusts) in a mosaic arrangement (Li et al., 2013). The reason for such heterogeneity is not entirely clear. Still, it seems to follow resource distribution in the soil and seems to be the result of an equilibrium with environmental stresses that prevents any LOD shift of the “poorer” biocrust patches (Li et al., 2016).

Moss crusts generally show improved catabolic rates and bring an alteration of microhabitats at the topsoil that establishes a vertical gradient for soil microbes and changes the niche overlap (Li et al., 2016). It has been observed that phototrophs drive the biodiversity of bacteria and fungi during biocrust development (Maier et al., 2018). Also, vascular plants can be more represented following biocrust onset, as different species naturally establish thanks to the improvement of soil stability and nutritional conditions (Colica et al., 2014; Lan et al., 2014; Wang et al., 2009; Zhao et al., 2014). It was observed how EPS content is higher at the topsoil after cyanobacterial inoculation compared to non-inoculated sand and soil (Colica et al., 2014). This is indicative of the higher availability of carbon sources readily accessible to the heterotrophic crust microflora (Chen et al., 2014; Mager and Thomas, 2010). Biocrust age and LOD are parameters that showed consistency with an increase in soil and sand enzymatic activity, moving from cyanobacterial crusts to lichen crusts (Miralles et al., 2012). Two classes of enzymes, hydrolase and sucrase, involved in the degradation of carbohydrates, are mainly directed to the more soluble smaller EPS fractions, which are thus readily utilizable by the heterotrophic microflora (Chen et al., 2014). The soil and sand EPS content is also consistent with an increase in water storage against evaporation and the quantity of water captured from non-rainfall sources (e.g., plant guttation, water vapor) (Adessi et al., 2018; Colica et al., 2014).

As opposed to optimal laboratory settings, under natural conditions it is impossible to control abiotic parameters; hence this may hinder moss growth, even after an initial establishment (Zhou et al., 2020). Therefore, inoculation in the field currently has the primary need of successful inoculation methodologies, evidencing and controlling the factors involved in moss establishment.

Some inoculation experiments in the field conducted in China using S. caninervis, B. argenteum, B. articum, and D. vinealis have been successful. However, it appears that one of the major constraints to large-scale inoculation for experimentalists is the actual low cultivation yields achieved in laboratories and greenhouses. The consequent low availability of inoculum sources has so far restricted the scale of outdoor moss application only to small plots (0.5 m2) (Zhou et al., 2020). At the same time, the inoculation of field-collected moss biocrusts has the drawback of needing the retrieval of intact biocrusts from close sacrificial areas, putting healthy areas at risk.

So far, both field inoculation studies conducted in China and in the Great Basin, USA, have enlightened the need to control watering, shade, and nutrient availability to attain moss establishment and growth in the field (Condon and Pyke, 2016; Zhou et al., 2020). For example, in China the maximal growth of B. argenteum to 70 stems per square centimeter was obtained in a 4-month period, by controlling nutrient availability, shade, and water provision (Bu et al., 2018). Similarly, Yang (2016) cultivated B. argenteum by inoculating propagules at a concentration of 500 g m-2 and watering at a rate of 3 L m-2 every 2 d until it reached a coverage of 70 % in 75 d (Yang, 2016). The application of a jute net, commonly used for erosion control, resulted functionally to increase water retention at the soil surface and sustain moss growth. The beneficial effects of the jute net probably also extend to lower soil temperature and exposure of mosses, keeping them more firmly in place (Condon and Pyke, 2016), increasing soil microtopography, and reducing wind and water erosion (Slate et al., 2020).