Introduction
National Aeronautics and Space Administration (NASA) has invested considerable resources (crops identification, growth chambers design, food processing equipment, among others) to guarantee fresh crops growth through bioregenerative food systems (BFS) for future missions to Mars (Perchonok et al. Reference Perchonok, Cooper and Catauro2012). Although, BFS were mainly focused on artificial growing medias (hydroponics, aeroponics, zeoponics, membrane systems), soil-based agriculture (SBA, i.e. using real soil growing media) has become increasingly relevant, achieving even higher productivity in some crops (Nelson et al. Reference Nelson, Dempster and Allen2008). Some authors (Silverstone et al. Reference Silverstone, Nelson, Alling and Allen2003; Kanazawa et al. Reference Kanazawa, Ishikawa, Tomita-Yokotani, Hashimoto, Kitaya, Yamashita, Nagatomo, Oshima and Wada2008; Maggi & Pallud Reference Maggi and Pallud2010) have pointed out that SBA using in-situ available resource of Martian surface is an important way to guarantee long-term sustainability for the future Martian colony. Mars today is a cold, dry desert world with surface conditions that are not habitable for even the hardiest known life forms from Earth (Davila et al. Reference Davila2010; McKay Reference McKay2010), however, there is evidence of past (or may be present) water activity and the presence of interesting niches for life (e.g., such as subsurface and/or evaporitic minerals) (Pottier et al. Reference Pottier, Forget, Navarro, Szantaib and Madeleine2017). Moreover, the Martian regolith is very salty and contains exotic salts such as sulphates and perchlorates (Hecht et al. Reference Hecht2009) becomes a major challenge for its use in agriculture (Wamelink et al. Reference Wamelink, Frissel, Krijnen, Verwoert and Goedhart2014). In this context, the use of terrestrial analogues of Martian surface constitutes an important effort to know and solve limitations to get SBA in the future (e.g. Silverstone et al. Reference Silverstone, Nelson, Alling and Allen2003, Reference Silverstone, Nelson, Alling and Allen2005; Kanazawa et al. Reference Kanazawa, Ishikawa, Tomita-Yokotani, Hashimoto, Kitaya, Yamashita, Nagatomo, Oshima and Wada2008; Nelson et al. Reference Nelson, Dempster and Allen2008). Mars-like soils on Earth provide a better understanding the physical, geochemical and microbiological processes that occur, or could have occurred, on Mars (Peters et al. Reference Peters, Abbey, Bearman, Mungas, Smith, Anderson, Douglas and Beegle2008; Valdivia-Silva et al. Reference Valdivia-Silva, Karouia, Navarro-González and Mckay2016). Appropriate soil's analogues on Earth are identified by their similar composition or environmental conditions that describe mechanisms that might guide the search for fossil and living evidence of microbial life (Preston & Dartnell Reference Preston and Dartnell2014) or/and simulate future problems if Martian soil will be used as a source of future crops and materials for human colonies (Bohle et al. Reference Bohle, Perez, Bille and Turnbull2016). An interesting Martian soil analogue studied and identified as a key analogue model for life in dry Mars-like conditions is Pampas de La Joya Desert located in southern Peru (Valdivia-Silva et al. Reference Valdivia-Silva, Navarro-González, Ortega-Gutierrez, Fletcher, Perez-Montaño, Condori-Apaza and McKay2011, Reference Valdivia-Silva, Karouia, Navarro-González and Mckay2016). The very low levels of organic carbon (10–40 ppm) and the presence of exotic minerals (including salts) and oxidants, could allow to identify and analyse the limits of growth in extreme conditions of different plants.
Potato is an extremely versatile crop with thousands of existing varieties adapted to grow well above the Arctic Circle to those able to grow in tropical regions, from 0 up to more than 4000 m above sea level including habitats with extreme weather and soil conditions (Zimmerer Reference Zimmerer1998; Birch et al. Reference Birch, Bryan, Fenton, Gilroy, Hein, Jones, Prashar, Taylor, Torrance and Toth2012). Wild relatives are found in even more extreme habitats, including extremely arid, saline and frost prone areas and can serve as a source of genetic traits for further adaptation (Martinez et al. Reference Martinez, Loureiro, Oliva and Maestri2001; Schafleitner et al. Reference Schafleitner2007; Vasquez-Robinet et al. Reference Vasquez-Robinet2008; Monneveux et al. Reference Monneveux, Ramírez and Pino2013). Potatoes are also extremely productive per unit of land area and water usage in comparison with most other staple crops (Renault & Wallender Reference Renault and Wallender2000) and are nutritious, rich in digestible starch, protein, fibres, vitamin C and B6, K, Mg and Fe (Woolfe Reference Woolfe1986). Therefore, the potato has been considered as a promising crop for growing in space exploration by NASA for many years (Perchonok et al. Reference Perchonok, Cooper and Catauro2012; Wheeler Reference Wheeler2017). An advanced population with wide genetic diversity and stable performance across divergent environments of the subtropical lowland agroecologies, resistance to main potato biotic and abiotic stresses, has been developed by International Potato Center (CIP) breeding program (CIP 2017). Such improved materials may prove their value beyond our planet to enable plant production in extreme environments of other planets. In this paper, it is reported a preliminary study testing a large and diverse panel of potato materials including native and improved varieties for their ability to grow and produce tubers in a Mars soil analog from La Joya desert in Southern Peru. The study aims were: – to analyse the limiting conditions imposed by the assessed soil – to identify potential materials with higher yield under the tested soil.
Materials and methods
Plant material
Sixty-five genotypes consisting of 38 advanced clones from the CIP Breeding Program for adaptation to subtropical lowlands and tolerance to abiotic stress, 22 native varieties from the taxonomic group Andigena, previously selected for drought tolerance (Cabello et al. Reference Cabello, De Mendiburu, Bonierbale, Monneveux, Roca and Chujoy2012) and five improved varieties (see Table 1) were chosen for this experiment. On 30 May 2016 six in vitro plantlets per genotype were transplanted to peat pellets (Jiffy Products Ltd., Canada), which were kept hydrated for 15 days until roots were well developed and plants reached 10–15 cm high.
Table 1. Advances clones (Adv Clone), improved varieties (Imp Variety) and Native potatoes tested in this study conserved in the International Potato Center (CIP) Gene Bank (see further details in CIP Catalogue, CIP 2017). Lowland tropical virus resistant (LTVR) breeding population. Surviving genotypes showed in Fig. 2 are remarked in grey
Soil sampling and characterization
The soil substrate was collected on 2 April 2016 from the hyper-arid area of Pampas de la Joya desert (quadrangle located between 16°38.386′ S–72°2.679′ W and 16°44.986′ S–71°58.279′ W), extensively studied for its geochemical Martian characteristics (Valdivia-Silva et al. Reference Valdivia-Silva, Navarro-González, Ortega-Gutierrez, Fletcher, Perez-Montaño, Condori-Apaza and McKay2011, Reference Valdivia-Silva, Navarro-González, Fletcher, Pérez-Montaño, Condori-Apaza, Ortega-Gutiérrez and McKay2012, Reference Valdivia-Silva, Karouia, Navarro-González and Mckay2016). This desert is the northern part of the Atacama Desert and is located to 50 km of the Arequipa city in Peru. To cover the spatial variability, approximately 700 kg of Mars-like soil was sampled from different points of the desert. The sampled soil was transported to CIP ‘La Molina’ experimental station located in Lima, Peru (12.08° S, 76.95° W, 244 m.a.s.l.) and a composite sample was analysed at Laboratorio de Suelo, Plantas, Aguas y Fertilizantes belonging to Universidad Agraria La Molina, Lima, Peru. The soil was loamy sand (72, 22 and 6% of sand, lime and clay, respectively) with very low organic matter (0.32%) and neutral pH (6.9 and 6.7 under 1 : 1 and saturation extract of the soil solution, respectively). The soil was hyper-saline (an electric conductivity of 19.3 and 52.6 dS m−1 under 1 : 1 and saturation extract of the soil solution, respectively) with a large prominence of Cl−1, Na1+and Mg2+ (580, 403.4 and 198.4 meq l−1, respectively) as soluble anions and cations.
Experimental conditions and management
On 27 June 2016 six peat-pellets with in-vitro plants of each genotype were transplanted in 1 l pots filled with Mars-like soil or a peat-based substrate (PRO-MIX, Premier Tech Horticulture, Canada), the latter serving as a control. All the pots were distributed in six plots randomly distributed in a greenhouse. Every plot had one plant of each genotype: three plots with a plant under Mars-like soil treatment and three plots with a plant under the control condition. All the pots were watered twice per week, to avoid soil leaching and therefore a fully assess for salt tolerance, the water quantity supplied was established through measurement of the maximum evapotranspiration per treatment through the gravimetric method every 2 weeks. For these ten randomly selected individuals per treatment were weighed before the irrigation and the target water quantity per soil treatment was defined as the maximum value estimated to recover the field capacity (see details of this method in Rolando et al. Reference Rolando, Ramírez, Yactayo, Monneveux and Quiroz2015). Based on soil analyses (see the previous sub-section) the fertilizer applications consisted of 200 : 100 : 240 : 20 mg kg−1 as N:P2O5 : K2O : CaO. In total, each pot was fertilized with 37.7 mg of Ca(NO3)2, 80.6 mg of NH4H2PO4, 159 mg of NH4.NO3 and 266.6 mg of KNO3, distributed in 2, 4, 8 and 6 weekly applications.
The trial duration was 134 days, under this period the average maximum and minimum daily temperature was 19.4 ± 0.2 and 15.2 ± 0.1 °C respectively and atmospheric humidity varied between 94.7 ± 0.3 and 72.0 ± 0.9% (atmospheric temperature and humidity sensor HC2S3 model, Campbell, USA). The daily average photosynthetic active radiation (PAR, 400–700 nm) was 2.60 ± 0.26, 3.05 ± 0.23, 4.78 ± 0.34 and 5.38 ± 0.29 MJ m−2 d−1 during July, August, September and October 2016, respectively (LI190SB model, LI-COR, USA). The daily global average atmospheric pressure was 984.3 ± 0.9 mb during July–October 2016 (PTB110 model, VAISALA, Finland).
Plant measurements
Physiological performance of plants under Mars-like condition in relation to the control was assessed through the mid-morning (taken from 8 to 10 am) or maximum light saturated (fixing 1200 µmol m−1 s−1 of PAR) stomatal conductance (g s_max; see details in Ramírez et al. Reference Ramírez2016) after and before two water pulses. For this purpose, four genotypes were chosen based on following criteria: (i) contrasted leaf chlorophyll concentration values in relation to the control plants (see formula (1)) assuming that plants with greener leaves in relation to the control were more affected by stress condition imposed by Mars-like soil (see Rolando et al. Reference Rolando, Ramírez, Yactayo, Monneveux and Quiroz2015); and (ii) plants with appropriate leaf size to be assessed in the cuvette of a portable photosynthesis system (LI-6400 TX, LICOR, Nebraska, USA). On 20 July 2016 leaf chlorophyll concentration (Chl SPAD) was assessed using a portable chlorophyll meter (SPAD-502 model, Konica Minolta, Japan), for this experiment four readings were taken of an apical leaflet belonging to a young, expanded and sun-exposed leaf and were averaged per plant. For each genotype Chl SPAD amplitude (Chl SPAD_Amp, proposed as stress tolerance index, Rolando et al. Reference Rolando, Ramírez, Yactayo, Monneveux and Quiroz2015) was estimated as follows:
$$Chl_{SPAD\_Amp} = X\; Chl_{SPAD\_MS} - \; X\; Chl_{SPAD\_c}$$Where X Chl SPAD_MS and X Chl SPAD_c were the Chl SPAD average value in the three plots under Mars-like and control soil treatments, respectively. Harvests (22 September and 8 November 2016) were performed when stems of plants grown in the control soil were brown and had fallen to the ground i.e. code 690 of senescence following Jefferies & Lawson's (Reference Jefferies and Lawson1991) classification. In the first harvest, some early genotypes and those that had already died in the Mars-like soil were sampled, whereas in the second harvest the majority of plants were in code 690 of senescence (i.e. ‘stems brown and fallen to the ground’). All the tubers were cleaned and weighted and among the surviving genotypes (established as those that survived and yielded in more than two plots) the percentage of fresh tuber yield (g plant−1) in relation to the control (% yield) was estimated as follows:
$$\% \; yield = \; \left( {\displaystyle{{X\; yield_{{\rm MS}}} \over {X\; yield_{\rm c}}}} \right)\; \% $$where X yield MS and X yield c were the average values of fresh tuber yield in plots under Mars-like soil and control treatments respectively.
Statistical analyses
Two-way ANOVA was performed to assess differences among genotypes, soil treatments and their interaction in fresh tuber yield. A linear regression between Chl SPAD_Amp and % yield was analysed in the surviving genotypes and the most influential points i.e. outliers with significantly affected in the regression line slope – were flagged using Cook's D and DFFITS tests (Rawlings Reference Rawlings1988). All the statistical analyses were run using R software (v. 3.3.3, R Core Team 2017).
Results
The selected genotypes for g s_max assessments showed Chl SPAD_Amp values of 9.3, 14.7, 15.1 and 19.9 corresponding to CIP 304350.18, CIP 388615.22, CIP 309043.123 and CIP 309068.7, respectively. Plants grown in control soil increased their g s_max to 82.7 ± 7.2 and 80.5 ± 17.1% on average after the first and second watering, respectively (Fig. 1). Potatoes in control soil, in particular after water pulses, showed g s_max > 150 mmol H2O m−2 s−1, whereas plants growing under Mars-like soil, showed g s_max < 50 mmol H2O m−2 s−1 (Fig. 1).
Fig. 1. Maximum stomatal conductance at saturating light (g s_max, mmol H2O m−2 s−1) assessment after and before irrigation pulses (discontinuous lines) in four genotypes (a: CIP 304350.18, b: CIP 309043.123, c: CIP 309068.7, d: CIP 388615.22) growing under standard (black circles) and Mars-like (open circles) soil conditions.
Forty percent of the assessed genotypes survived under Mars-like soil condition with a fresh tuber yield ranging between 0.3 and 5.2 g plant−1 (Fig. 2(a)). The 2-way ANOVA detected significant differences in soil types (F = 541.0, P = 0.048), genotypes (F = 3.9, P < 0.001) and their interaction (F = 4.7, P = 0.031). The % yield as compared with control soil was ranged between 0.3 and 9.3%, being CIP 397099.4, CIP 396311.1 and ‘Tacna’ variety (CIP 390478.9) the genotypes with the highest values (9.3, 8.9 and 5.8%, respectively; Fig. 2(b)). The fitted linear function between Chl SPAD_Amp versus % yield showed a negative slope (y = 19.3 − 1.1x; R 2 = 0.25) (Fig. 3). The more influent points were the ordinate pairs [x;y]: [1.6%;28.3], [8.9%;5.3] and [9.3%;10.7] defined by Cook's D (0.17, 0.24 and 0.09, respectively) and DFFITS (0.65, −0.70 and 0.43, respectively) tests (Fig. 3).
Fig. 2. Fresh tuber yield of the survivor potatoes genotypes growing in Mars-like soil condition expressed as average fresh tuber yield (a) and – average percentage of tuber yield in relation to the yield under the standard soil (b).
Fig. 3. Scatter plot of the average percentage of fresh tuber yield in potatoes genotypes growing in Mars-like soil in relation to the yield under the standard soil (% yield) versus difference of the average chlorophyll SPAD values under Mars-like soil and average chlorophyll SPAD values under the standard soil (Chl SPAD_Amp, without units) measured on 21 July 2016. In grey the more influent points defined by Cook's D and DFFITS tests.
Discussion
Physiological performance and tuber yield under Mars-like soil condition
In particular, after water pulses, potatoes in control soil showed g s_max > 150 mmol H2O m−2 s−1 (Fig. 1), which has been identified as an appropriate indicator for optimum irrigation and where plants are under optimum conditions (Flexas et al. Reference Flexas, Bota, Loreto, Cornic and Sharkey2004). On the other hand, plants growing under Mars-like soil and even after water pulses showed g s_max < 50 mmol H2O m−2 s−1, which is defined as a physiological severity threshold in potato (Ramírez et al. Reference Ramírez2016) where plants are likely submitted to irreversible physiological (oxidative) damage Medrano et al. (Reference Medrano, Escalona, Bota and Flexas2002). This last result and the low tuber yield in relation to the control (Fig. 2) confirms the difficult growing condition characterized by an extremely high soil salinity (see the section Materials and Methods) far beyond that tested in any other studies (from 2.3 to 16.2 dS m−1 of electric conductivity) looking for salt effect in potato (see Katerji et al. Reference Katerji, van Hoorn, Hamdy and Mastrorilli2000; Shaterian et al. Reference Shaterian, Waterer, Jong and Tanino2005; Nagaz et al. Reference Nagaz, Masmoudi and Mechlia2007). Salts dominated by sulphates, carbonates, chlorides and nitrates are identified as important likely components of Mars regolith (Clark & Van Hart Reference Clark and Van Hart1981; Osterloo et al. Reference Osterloo, Hamilton, Bandfield, Glotch, Baldridge, Christensen, Tornabene and Anderson2008), so extreme salinity conditions such as Mars regolith pose potential problem to grow crops in for future SBA missions (Silverstone et al. Reference Silverstone, Nelson, Alling and Allen2003; Ewing et al. Reference Ewing, Sutter, Owen, Nishiizumi, Sharp, Cliff, Perry, Dietrich, McKay and Amundson2006). It is necessary to design methods to remove or reduce salinity toxicity (e.g. testing previous leaching treatments) but also improve the fertility level of Martian regolith through the incorporation of organic matter recycled from solid waste composting activities from the human habitat (Silverstone et al. Reference Silverstone, Nelson, Alling and Allen2003; Nelson et al. Reference Nelson, Dempster and Allen2008). The use of microorganisms to degrade organic matter (Kanazawa et al. Reference Kanazawa, Ishikawa, Tomita-Yokotani, Hashimoto, Kitaya, Yamashita, Nagatomo, Oshima and Wada2008) and process remnant salt components (Matsubara et al. Reference Matsubara, Fujishima, Saltikov, Nakamura and Rothschild2017), including nanoparticles for soil remediation (Patra et al. Reference Patra, Adhikari, Bhardwaj, Dagar, Sharma, Sharma and Singh2016), will be important for a sustainable SBA in Mars. Indeed, Martian regolith has high presence of different types of salts and evaporitic minerals i.e. formed by the evaporation from bodies of water (Vaniman et al. Reference Vaniman, Bish, Chipera, Fialips, Carey and Feldman2004; Ewing et al. Reference Ewing, Sutter, Owen, Nishiizumi, Sharp, Cliff, Perry, Dietrich, McKay and Amundson2006) and they have been detected on Mars both in situ and remotely by different monitoring instruments (Wadsworth & Cockell Reference Wadsworth and Cockell2017). The controversy about their effects on the habitability of that planet is still under research. Thus, some studies have positive implications showing these minerals as possible electron acceptors by microorganisms capable to provide energy for growth or as powerful antioxidants protecting plants against Mars’ harsh environmental stresses and boost the rate of decomposition of organic matter (Bohle et al. Reference Bohle, Perez, Bille and Turnbull2016). On the contrary, other studies show the Martian salts as a detrimental condition for life survival (Wadsworth & Cockell Reference Wadsworth and Cockell2017). The presence of different living beings in extreme salt condition on Earth such as the halophilic organisms encourage the options to generate future crops and better understand the mechanisms of survival in these conditions.
Despite the extreme salinity, 40% of the genotypes survived (Fig. 2). There is a debate if potato is considered as salt sensitive (Maas & Hoffman Reference Maas and Hoffman1997; Larcher Reference Larcher2003; Nagaz et al. Reference Nagaz, Masmoudi and Mechlia2007; Levy et al. Reference Levy, Coleman and Veilleux2013) or tolerant (Katerji et al. Reference Katerji, van Hoorn, Hamdy and Mastrorilli2000). However, whatever the classification, models estimated as the slope of % yield reduction versus soil electrical conductivity in previous studies (−5.6, −12 and from −34 to −54%/dS m−1 corresponding to Maas & Hoffman Reference Maas and Hoffman1997; Katerji et al. Reference Katerji, van Hoorn, Hamdy and Mastrorilli2000 and Nagaz et al. Reference Nagaz, Masmoudi and Mechlia2007, respectively) predict no tuber yield under the salt levels found in the Mars-like soil used in this study. In contrast to these predictions, there was tuber yield as compared the control soil (0.3–9.3%; Fig. 2(b)) highlighting the potential of the assessed genetic material to produce under extreme saline conditions, meriting further studies.
Promising tolerant genotypes and physiological indicators for extreme salinity
CIP 397099.4 and CIP 396311.1, which are advanced clones belonging to CIP lowland tropic virus resistant breeding population (CIP 2017), were identified as the most tolerant to the Mars-like soil with % yield >8% compared with the control (Fig. 2(b)). CIP 396311.1 is an advanced clone with extreme resistance to PVY an PVX, early maturing and tolerant to heat has shown good yields in sites affected by high soil salinity in Southern Bangladesh (Amoros personal communication). The ‘Tacna’ variety (CIP 390478.9) a genotype also with extreme resistance to PVY and PVX, selected from arid and saline environments of the Southern Peruvian Coast (Zegarra & Fernández Reference Zegarra and Fernández2013) showed a yield >5% compared with the control (Fig. 2(b)). This variety is considered as drought and heat tolerant (CIP 2017) with high yields under water restriction conditions reported in Uzbekistan (Carli et al. Reference Carli, Yuldashev, Khalikov, Condori, Mares and Monneveux2014) and China (locally named as ‘Jizhangshu 8′; He et al. Reference He, Chen, Gui, Wang and Huang2013; Wang et al. Reference Wang, Wang and Wang2014). Because some mechanisms of resistance are unspecific to the kind of stressors (Larcher Reference Larcher2003), it is expected that genotypes highly resistant to biotic (virus PVY and PVX) and other abiotic (drought and heat) stresses, could also show tolerance to other unreported stressors like salinity. Drought and salinity tolerance share common physiological mechanism (Chaves et al. Reference Chaves, Flexas and Pinheiro2009), so it is expected that some of the traits selected by phenotyping for drought tolerance could confer resistance to salinity also. This was supported by the inverse relationship found between Chl SPAD_Amp and % yield (Fig. 3) predicted by Rolando et al. (Reference Rolando, Ramírez, Yactayo, Monneveux and Quiroz2015) under drought stress. Potatoes leaves under stress reduce their growth, concentrating their chlorophyll in less area and appear greener when they are more sensitive to drought (Ramírez et al. Reference Ramírez, Yactayo, Gutiérrez, Mares, De Mendiburu, Posadas and Quiroz2014; Rolando et al. Reference Rolando, Ramírez, Yactayo, Monneveux and Quiroz2015). Although the predicting capacity of the fitted function was slight (R 2 = 0.25), the more influent points were those that showed the higher and lower Chl SPAD_Amp values (Fig. 3), the latter of which corresponded to the genotypes with higher tolerance to Mars-like soil mentioned above (CIP 397099.4 and CIP 396311.1). Greenness inspection through Chl SPAD_Amp may, therefore, be a worthwhile predictor of high tolerant genotypes under extreme salinity that could be used in future breeding programs.
Conclusion
Extreme soil salinity will be an important stressor to the growth of any plants using Martian soil. Under a controlled/protected environment with pressurized atmosphere, a combination of an appropriate sowing method, tolerant genotypes and soil management will be crucial to achieve yield in such conditions. In this preliminary study, In vitro plantlets of two advance clones (CIP 397099.4 and CIP 396311.1) rooted in peat pellets substratum and transplanted into Mars-like soil under drip irrigation, were able to yield more than 8% of tuber biomass as compared with the control under the highest salinity condition reported in scientific studies for potatoes. More studies are necessary to increase the yield in these genotypes through long-stress memory improvement (see Ramírez et al. Reference Ramírez, Rolando, Yactayo, Monneveux, Mares and Quiroz2015), to test appropriate controlled atmospheric conditions and soil treatments to reduce extreme salinity effects with a concomitant increase of water and nutrients availability.
Acknowledgements
The financial support for this experiment was provided by The International Potato Center. The authors thank the support of Nikolai Alarcon, Walter Gomez, Paulo Garcia, Jesus Zamalloa and Javier Rinza. MSc. Felipe De Mendiburu helped us in the statistical analyses design.
Open access
