The production of the Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] Christmas tree in forest plantations located in Valle de Bravo, State of Mexico, Mexico, requires yearly edaphic and foliar fertilization with a balance: 5N:1P:1K to enhance the growth of the new sprouts. Although these sprouts and all the needles of the foliage are vulnerable to Phaeocryptopus gaeumannii (Rhode) Petrak. An Ascomycota (^{Shaw et al., 2011}) that grow intercellularly in the needles (^{Stone et al., 2008}), and forms black globose pseudothecia that align on the stomata on the underside (^{Dennis, 1978}), and induces severe defoliation (^{Skilling, 1981}). Fertilization has been used to control the disease. The excess N increases the severity of Swiss needle cast (^{El-Hajj et al., 2004}), and balanced with P, it does not increase it (^{Mulvey et al., 2013}); and healthy needles in proportion to N have low P doses (^{Mohren et al., 1986}). Silicon (Si) has not been used to control Swiss needle cast. In other conifers, it is thought to help reduce toxicity by aluminum (^{Hodson and Sangster, 1999}). Si is not included in nutrient formulations since it is not considered essential. Plants accumulate it naturally in the intercellular spaces, in the cell wall, the cell lumen, epidermis, and cuticle; and it possible acts as an adverse factor in the adequate nutrition of fungi. This is based on the fact that in diverse crops it contributes to the reduction of diseases (^{Datnoff et al., 2011}). Based on the general concepts by ^{Griffin (1994)} and ^{Jennings (2007)} on fungal nutrition and mycelial growth, we can extrapolate that, when Si adheres to the cell wall, the hyphal enzymes do not act efficiently when unfolding cellulose into glucose, and fungal nutrition is affected negatively, reducing the disease (^{Datnoff et al., 2011}). In Mexico this disease has been found in 11 states, in populations of wild P. menziesii (^{Cibrián et al., 2007}; ^{Cibrián et al., 2014}), and in 2005, in the region studied, with severe defoliation, and its control is only chemical (^{Stone et al., 2007}). The aim of the study was to evaluate in vitro the effect of different macronutrientes and micronutrients and the Si on the growth in vitro of P. gaeumannii. We hypothesized that the macronutrients and micronutrients added to the culture medium affect the fungal growth, that Si is toxic, and in the presence of Si the fungus cannot properly absorb the sources of carbon of the culture medium, reducing its hyphal growth rate.

The study was carried out with an isolation of P. gaeumannii (# JN204508, NCBI) from the Culture Collection of the Colegio de PostgraduadosPlant Pathology (# CP-A032), isolated from P. menziesii Christmas trees in Valle de Bravo. The culture was increased in a 2 % Malt Extract Agar (MEA) (^{Crous et al., 2009}) (Oxoid LTD, LM0059, England) at a constant temperature of 21 °C and complete darkness for 20 days. To compare the growth responses and as a double evaluation in time, two in vitro experiments were conducted. One, in a solid medium (2 % MEA), and the other, in a liquid Potato Dextrose (PD) medium.

First experiment. In a factorial experiment with a completely random design, five repetitions and a total of 50 experimental units, ten treatments were evaluated (Table 1). The macronutrient solution had the nutritional balance of 3N: 1P: 3K (100 mL^-1 per treatment), and sodium metasilicate (44 % Si; Na₂SiO₃) as a source of Si. The micronutrients were prepared and applied as described by ^{Hewitt and Smith (1975)}. To the solutions (Table 1) were added 2 % MEA, sterilized them at 120 °C for 20 min, it was measured the pH and adjusted it to 5.5 with 0.01N H₂SO₄, and pour the medium into sterilized plastic Petri dishes with 8.5 cm in diameter (diam.). For each dish, 1 mm fragment of the 20 day old P. gaeumannii culture were plated and incubated at a constant temperature of 21 °C and total darkness for 42 days. The growth of the diam. of the culture (mm) was measured every seven days and the data were analyzed using ^{Fisher’s growth analysis technique (1921)}. The data were converted into a natural logarithm (Ln) and a linear regression was carried out (^{Hunt, 2003}). The Instant Relative Growth Rate (R or iRGR) was obtained with the following model: Ln a = b + R (DDIE) [where: a = variable evaluated, b = value where the straight line crosses the ordinate, R = iRGR and DABE = Days After the Beginning of the Experiment]. The iRGR of the control was subtracted from each treatment and the data are shown in Figure 1A. The values above (+) the “x” axis were the differences in which each treatment surpassed the iRGR of the control, and the values below (-) the “x” axis were the differences in which each treatment fell below the iRGR of the control.

Table 1. Nutrients and doses of silicon used in two in vitro experiments in a solid medium 2 % of MEA and liquid PD to determine the growth of the colony (cm) and the dry weight (mg) of the mycelium of Phaeocryptopus gaeumannii. 

^wMalt extract Agar; ^xPotato Dextrose; ^yFormulation in text; ^{zHewitt and Smith, 1975}.

Figure 1. Growth of P. gaeumannii in solid and liquid media supplemented with different nutrients and doses of silicon. A) Relative Growth Rate (iRGR) in a solid medium of 2 % Malt Extract Agar for 42 days, B) Relative growth (mg) in liquid Potato Dextrose medium determined after 35 days. Bars above the “x” axis (+) indicated that the difference in growth of the treatment was higher in relation to the control, and below the axis (-), it was lower in relation to the control. Macros = macronutrients, Micros = micronutrients, Si = Silicon. 

Second experiment. This was carried out in a completely random design with five repetitions, 21 treatments (11-31) (Table 1), and a total of 105 experimental units. The control (T31) was only the liquid medium of Potato Dextrose [PD; broth: 200 g white potato without the peel, cut into small pieces and boiled for 20 min in distilled water, and 14 g dextrose (J.T. Baker, 1916-01, Mexico) per liter of distilled water] plus the fungus. Into the medium was added the same nutrient solution mentioned above, some doses changed (Table 1), and an incubation period for 35 days. The colony developed in each tube was extracted from the liquid medium, placed in sterilized aluminum foil, and the humidet weight was taken on an electronic scale with an approximation of 0.001 g (AP210- O, Ohaus, New Jersey, U.S.A.). The mycelium samples were transferred to crystal (Pyrex) 5.0 cm Petri dishes and dehydrated in an oven (Craft Instrumentos Científicos, Mexico) at 35 °C for 48 h and the dry weight was taken. For each humid and dry weight variable, an analysis of variance was carried out and a Tukey comparison of averages (α =0.05) using the statistical package SAS (2009) version 9.3 (SAS The Power to Know, USA). Each dry weight average for the treatments was subtracted the average of the control. Data in Figure 1B.

The results of both experiments were statistically significant.

First experiment. In all treatments there was growth of the P. gaeumannii colony (Figure 1A). The macronutrients (T1) alone favored the iRGR, although, when the micronutrients were added (T7) the growth stimulus increased, as expected in a balanced formulation. On the other hand, when there were only micronutrients in the medium (T4) iRGR decreased. In the presence of macronutrients (T1 and T7) the fungus obtained N and P, basic elements for the formation of protein substances and secondary metabolites that contribute to the growth of the fungus. The negative effect of the microelements (T4) was reduced in the presence of macroelements (T7) which promoted a nutritional balance for the iRGR. On the other hand, the addition of Si in the other treatments reduced the iRGR (Figure 1A). The greatest reduction in the growth of the colony was observed when the medium contained micronutrients (T5) and 250 ppm of Si (Figure 1A). The highest concentration of Si (1000 ppm) (T6), did not surpass the inhibitory effect due to the combination micronutrients - 250 ppm Si (T5). This leads to infer that the concentration of the microelements must be balanced with that of Si for an effective inhibition of fungal growth. The inhibitory effect of the Si was corroborated in the treatment in which macronutrients were added to the medium, along with micronutrients plus 1000 ppm of Si (T9). Since there are no reports in the bibliography on the inhibitory effect of Si on the growth of fungi in vitro, our hypothesis is that this could be a toxicity response. Also, if we consider that in plant nutrition (including P. menziesii Christmas trees) a balanced formula is required between macronutrients and micronutrients, studies on treatment T9 should continue. Although treatment T5 was the best of all, the micronutrients on their own do not nourish plants, yet it indicated that their concentration was key in the reduction of fungal iRGR.

Second experiment. Results were based on the dry weight (there were no significant differences with the humid weight). The treatments that best stimulated the mycelial growth of P. gaeumannii were seven (bars above the “x” axis), and where the treatments with double concentrations of macronutrients with or without Si (T14, T15) stood out (Figure 1B). This, as mentioned earlier, could have been due to the N and P that favored a greater mycelial growth, although in normal doses, its effect was negative (T11). This could be due to the fact that in a solid medium (T1) hyphae had a greater growth efficiency because it is an aerobic fungus (^{Griffin, 1994}), and in the liquid medium, treatment T14 required twice as many macronutrients to favor the growth in an adverse condition. Treatment T30 with only high doses of Si also promoted fungal growth although in lower amounts. This is possibly due to the nutrients of the liquid medium such as starches (N) and traces of protein derived from the potato extract (PD). On the contrary, in low doses (T29), Si inhibited fungal growth. Both results must continue to be studied using a treatment with water and Si alone. It would also be recommendable to use other Si sources such as Silica Gel, NaSiO3 (^{Griffin, 1994}), etc. The other 14 treatments displayed an inhibitory effect in fungal growth (bars below the “x” axis) (-). In relation to the complete micronutrients (Table 1; six elements) in normal doses and with or without Si they behaved as in the first experiment, and also in a double dose (T22, T23), and restricted fungal growth. When reducing micronutrients to three elements (Table 1; Cu, Mn, Zn), treatment T18 behaved the same as T19 (six elements), which led to infer that these three micronutrients could be the ones that inhibited mycelial growth, and Mn in particular. ^{Griffin (1994)} pointed out that the excess Mn, as in treatment T18, acts by inhibiting fungal growth. However, when adding Si, fungal growth was not affected (T17; normal dose) and growth was similar to the control. More experiments should be designed with double doses of micronutrients + Si. Macronutrients plus micronutrients in double doses, and with and without Si, stimulated mycelial growth (causes mentioned above); and in normal doses, they restricted it (Figure 1). The treatment that stood out was T25 with the highest mycelial inhibition. The balanced formulation of macroelements and microelements expected in the nutrition of plants, which also included Si as a coadjuvant, indicate that T25 is the best treatment in the liquid medium, which was also observed for treatment T9 in the solid medium. In this way, considering that Si may contribute to reduce the disease (^{Datnoff et al., 2011}), and in complementing an adequate fertilization formulation, it would help to balance the nutritional content of the plant (^{Mohren et al., 1986}), the possibilities of controlling Swiss needle cast could be feasible in future studies. In conclusion, this is the first study on the effect of Si and micronutrients Cu, Mn, Zn on the inhibition of the growth in vitro of the fungus P. gaeumanni, causal agent of P. menziesii in Christmas tree plantations.