Plant material

Strawberry plants (Fragaria × ananassa Duch.) cv. Festival from a commercial nursery in central Mexico were planted on August 26, 2016 inside a polyethylene macro-tunnel (34.0 m length, 5.0 m width, 2.9 m height) located at 19^o 20’ N, 98^o 53’ W and 2250 masl. Temperature records inside the tunnel are shown in Figure 1. Plants were chilled at 4 ^oC for one month and then planted on beds (20.0 m length, 1.1 m width, 0.35 m height) filled with either red volcanic rock or local agricultural soil. Beds were previously disinfected with Metalaxil-M for soil-borne diseases control following commercial recommendations. There were three rows of plants per bed with 30 cm spacing between rows and 20 or 30 cm between plants, resulting in plant densities of 15 and 10 plants m^-2, respectively.

Figure 1 Temperature inside the plastic macro-tunnel. Highlighted section shows the temperature used for the growing degree days (GDD) computation. Dotted central line represents the average total temperature during the experiment; the arrow shows the planting date. 

Cultivation system

A Steiner mineral solution ^{(Steiner, 1984)} adjusted to 50 % of its original concentration, pH 5.5 to 6.0 and electric conductivity (EC) of 1.2 to 2.0 dS m^-1 was used in a fertigation system with three drip lines and compensated emitters (3.5 mL min^-1) at 20 cm separation. The system was set to allow five events per day from 9:00 am to 6:00 pm for a total of 16 min dripping for the red volcanic rock substrate and one event of 10-12 min every other day at 11:00 am for the soil. Volcanic red rock was < 1.0 cm particle diameter and came from local mines; physical and chemical properties for this substrate have been reported by ^{Trejo-Tellez et al. (2013)}. Local agricultural soil was clay loam with pH 7.5, EC 0.5 dS m^-1, bulk density 1.23 g cm^-3 and CaCO₃ 15.8 %. Pests and weeds were controlled following commercial recommendations for strawberry.

Phenological stages

Temperatures were recorded every hour from the day of planting by using a sensor placed 50 cm above ground level and connected to a Dataloguer (HOBO^®, Onset Computer Corporation, Bourne, Massachusetts, USA). Daily growing degree days (GDD) after planting date were computed with temperature data and the cumulative GDD requirements for phenological stages, as decribed by ^{Enz and Dachler (1997)} for strawberry, were determined: early balloon stage (first flowers with petals forming a hollow ball), onset of anthesis (first flowers open), flowers fading (majority of petals fallen), receptacle protruding from sepal whorl, seed clearly visible on receptacle tissue, beginning of ripening (most fruits white in color), first fruits with cultivar-specific color. Daily GDD were computed using the single sine method (Equation 1) with lower and upper threshold temperatures of 3.0 and 30 °C, respectively ^{(Elnesr and Alazba, 2016}; ^{Krüger et al., 2012};), fitting inside the range every temperature found outside the thresholds.

Where GDD: daily growing degree days, Tmax: maximum temperature for the day, Tmin: minimum temperature for the day, Tbase: 3 °C.

Experimental design and statistical analysis

The experiment was conducted under a completely randomized design and split-plot arrangement with factor substrate (red volcanic rock and soil) as the main plots and factor plant density (10 or 15 plants m^-2) as the split plot with three replications (beds). Distribution of treatment resulted from dividing three beds corresponding to each substrate into two equal sections and randomly assigning plant density to each section. Twenty plants from the central part of each replication were labeled and used to collect data. Resulting data were submitted to analysis of variance by using the GLM procedure of the SAS v9.0 program and mean separation by the LSD test (P ≤ 0.05) to compare treatment means. Linear regression was performed to predict the phyllochron and the DAP in every phase of reproductive phenology using the GDD. Prior to the regression analysis, natural logarithm transformation of the independent variable was performed to estimate the best linear fit between the involved variables ^{(Ott and Longnecker, 2001)}.

Growing degree days (GDD) vs. days after planting (DAP)

High correlation coefficients (r > 0.964) between GDD and DAP were found for each phenological stage considered in this study (Table 1). Confidence interval (95 % confidence) for the slope was under a 20-day range. The decision to choose one or the other method to count the time to reach a specific phenological phase will depend on the benefits of each one. ^{Parra-Coronado et al. (2015)} found that GDD can reliably predict the days to the onset of various phenological stages in feijoa [Acca sellowiana (O. Berg) Burret] grown in different natural environments. Chronology to beginning of the phenological phases of cultivated fruit species has traditionally been reported in calendar days; however, because of the influence of temperature on plant phenology, these values could vary significantly for the same cultivar grown in different environments. In contrast, GDD-based phenology becomes important in determininig the onset of phenological stages by providing a more reliable tool in scheduling activities in a specific crop cycle ^{(Krüger et al., 2012}; ^{Parra-Coronado et al., 2015}; ^{Parthasarathi et al., 2013}; ^{Rahman et al., 2016)}.

Leafing phenology (Phyllochron)

The analysis of this process becomes relevant since the productivity of the Festival strawberry is highly correlated with its foliar development ^{(Menzel and Smith, 2014)} and the data from these type of analysis might be used to generate models that facilitate detailed scheduling of activities, thus increasing productivity. In this experiment, leaf formation was not different between treatments up to 163 DAP (February 4, 2017); afterwards, plants grown in soil formed more leaves than those grown on red volcanic rock (Figure 2). Compared to soil, red volcanic rock has low water retention and low water use efficiency (fruit mass per volume of water) ^{(Ojodeagua et al., 2008}; ^{Trejo-Téllez et al., 2013)}. It seems that water availability was not a limiting factor for plants of any treatment during most of the growing season; however, during the last third of the season, as the plant accumulated more leaves, the water on red volcanic rock was clearly insufficient to maintain the same leafing rate compared to the soil, even though there were no obvious symptoms of water stress in plants of both substrates throughout the growing season. During the experiment, plant density had no effect on the number of leaves per plant and there was interaction between factors (substrate × plant density; P = 0.003) only on the last sampling date (March 22, 2017) resulting in more leaves (12.9) in the plants grown in soil at low plant density (10 plants m^-2).

Figure 2 Leafing in Festival strawberry plants grown in soil or red volcanic rock in a plastic macro-tunnel in central Mexico. Difference between means is non-significant (NS), significant (*) or highly significant (**) according to the LSD test. Bars are the standard error of the mean (n = 6). 

Treatments had no significant effect on phyllochron in this experiment (Figure 3A). There was a requirement of 597 GDD for the onset of leafing and the second leaf to be evident. Other studies have demonstrated that phyllochron in strawberry may depend on the cultivar, the origin of the plant and the planting date or the interaction among these factors ^{(Rosa et al., 2011}; ^{Tazzo et al., 2015}; ^{Thiesen et al., 2018)}; furthermore, the chilling treatment to the crown before planting may or may not have a significant effect on this variable ^{(da Costa et al., 2014}; ^{Diel et al., 2017b)}; nevertheless, all these studies agreed on a high correspondence between air temperature and phyllochron, which was also found in this experiment (R² = 0.9754; Figure 3B). All these previous studies indicate that every period between leaves in the strawberry crown is highly genetically influenced yet significantly modulated by air temperature; in addition, crown carbohydrates, either stored or newly synthesized, are important for this modulation ^{(da Costa et al., 2014}; ^{Rosa et al., 2011)}. Though the strawberry phyllochron and all the regulating factors should be carefully investigated, this experiment shows that despite the fact that cultivation on red volcanic rock could decrease the final number of leaves on the strawberry crown compared with cultivation in soil, its phyllochron based on GDD remains relatively constant.

Figure 3 A) Growing degree days (GDD) to consecutive leaves formation in the crown (phyllochron), B) linear regression to predict leaf number from natural logarithm of GDD. Bars in A are the standard error of the mean (n = 3). 

Reproductive phenology

All phenological phases studied in this research are also considered in the BBCH code for strawberry ^{(Enz and Dachler, 1997)}. Literature on strawberry phenology that considers the BBCH code and that is based on GDD is scarce. Data from this experiment are in the range reported in literature for strawberry Elsanta, Korona and Clery grown at different latitudes in Europe, from Italy to Norway, by ^{Krüger et al. (2012)}, either for DAP to the onset of flowering and harvest (red fruit) or for GDD accumulated during the period between these two stages (Table 2). Strawberry reproductive phenology is genetically regulated and environmentally modulated by temperature, photoperiod, humidity, and plant nutrition ^{(Diel et al., 2017a}; ^{Krüger et al., 2012}; ^{Rahman et al., 2016}; ^{Sønsteby and Heide, 2006}; ^{Wan et al., 2018)}; as a result, activities at the place of origin of the plantlets and during their cultivation could modify the reproductive phenology and the phyllochron while growing in substrates under plastic cover ^{(Diel et al., 2017a}; ^2017b). In this study there was no significant effect of the substrate (soil vs. red volcanic rock) on reproductive phenology of Festival strawberry, meaning that plants had equivalent conditions before and after planting or at least not significant enough to generate differences between treatments for most of the registered variables.

Regarding plant density, there was a significant delay of 17 days to reach the early balloon stage and the onset of anthesis in plants at high density (15 plants m^-2) compared to plants at low density (10 plants m^-2), but this difference disappeared thereafter (Table 2). Nitrogen is a mineral that can affect floral differentiation in strawberry ^{(Wan et al., 2018)}, and when applied three weeks after the beginning of short-day conditions it can advance flowering by eight days ^{(Woznicki et al., 2018)}. The plants in this experiment were exposed to short days just one month after the planting date; thus, plants at high densities could have a stronger competition for nitrogen than those at low densities, which affects the first two reproductive stages considered in this study. The lack of effect of plant density on the later reproductive stages is probably due to the similar foliar development between treatments during most of the growing period and to the similar de novo synthesis of carbohydrate which apparently is determinant for reproductive phenology in strawberry ^{(Menzel and Smith, 2014)}.