Eucalyptus Field Growth and Colonization of Clones Pre-Inoculated with Ectomycorrhizal Fungi

Abstract

Ectomycorrhizae are classified as biotechnology to increase the sustainability of planted forests, and fieldwork is needed to confirm its effectiveness. The growth of rooted cuttings of Eucalyptus clones GG100 and GG680, which had been previously inoculated in the nursery with ectomycorrhizal fungi, was evaluated after planting them in the field. The ectomycorrhizal fungi (EMF) inoculated were: Pisolithus microcarpus, Hysterangium gardneri, or Scleroderma areolatum. Uninoculated rooted cuttings were used as controls. The inoculated treatments and the uninoculated controls (low P control) were grown in a nursery with reduced phosphate fertilization. Additionally, uninoculated controls were grown on a substrate with complete phosphate nursery fertilization (high P control). After two months, the plant height of clone GG100 inoculated with P. microcarpus was 16% taller and of clone GG680 13% higher than the low P control treatment. At the same time, the collar diameter of the plants inoculated with H. gardneri and P. microcarpus was the same as in the high P control. At 12 months, the growth of the inoculated and low P control plants was the same as in the high P control. For ectomycorrhizal colonization, after six months, the mean percentage of colonized root tips was highest in plants inoculated with S. areolatum, followed by those inoculated with P. microcarpus and of the low P control. After one year, ECM colonization was equal in all treatments and 4.3 times greater than it was at 6 months. Inoculation with ECM fungi in the nursery boosts early plant growth after transfer to the field, although the effect depends on the specific ectomycorrhizal fungus and the clone. Further ectomycorrhizal colonization of Eucalyptus occurs naturally and increases with tree development in the field.

1. Introduction

Eucalyptus is one of the most used plants to produce coal, cellulose, and paper in the world [1]. The success of Eucalyptus stands as a result of the species’ high adaptability to soils with limited phosphorus (P) and nitrogen (N) levels [2] and of the association of its root system with ectomycorrhizal fungi (EMF), forming ectomycorrhizae. This mutual association enables plants to increase the soil volume explored by the roots and, consequently, the quantities of nutrient and water uptake [3,4,5]. These fungi contribute to tree nutrition by nutrient mobilization from organic matter and minerals via enzyme secretion and mineral weathering; in exchange, the fungi are supplied with photo-assimilates produced by the trees [6]. The ectomycorrhizal formations contribute effectively to P, N, Calcium (Ca), Potassium (K), Magnesium (Mg), and micronutrient uptake by the host plant, due to the smaller diameter of the hyphae, which can explore tiny pores inaccessible to the roots and producing and releasing appropriate enzymes that act to mineralize organic sources of nutrients or solubilize mineral sources [7,8,9,10,11,12]. This is important, as a recent survey estimated that 61% of the natural terrestrial land area is significantly limited by N or P; and the remaining 39% of the natural terrestrial land area could be co-limited by N and P or weakly limited by either nutrient alone [13].

In Eucalyptus, in vitro inoculation with Pisolithus sp. increased seedling growth by 34.6% [14]. In the greenhouse, Pisolithus microcarpus isolate UFSC-Pt116 increased root dry mass, shoot dry mass, and height and diameter of the stem [15,16,17]. In a commercial nursery, isolates increased the growth of inoculated hybrid cuttings by 13.1% [12]. In commercial Eucalyptus plantations, especially in Latin America, knowledge about ectomycorrhizal colonization is poorly known when compared to other regions of the world and with other tree species. Other studies with different species reported that, in the field, growth increases when inoculated seedlings were used for out-planting. For example, Pinus radiata inoculated with Rhizopogon roseolus and Scleroderma citrinum [18] and Pinus pinea inoculated with Rhizopogon luteolus and R. roseolus [19] in the seedling stage survived and grew better than uninoculated seedlings after planting in the field. The EMFs Laccaria bicolor, Melanogaster ambiguus, Rhizopogon clossus, Rhizopogon subareolatus inoculated on Douglas-fir [20], and Pisolithus tinctorius inoculated on Pinus [21], persisted for many years in the field and intensified tree survival and growth.

Thus, the establishment of EMF-colonized forest plantations presents an alternative to fortify the sustainability of planted forests and reduce the dependence on chemical fertilizers from non-renewable sources. The inoculation of nursery seedlings could introduce previously selected fungus to the field with high chances of facilitating an adequate plant establishment and development. However, the degree of response to ectomycorrhizal inoculation in reforested areas depends on the colonization state of seedlings at planting, the planting site, persistence of the introduced fungus, and other local biotic and abiotic factors [18,22,23]. Therefore, the inoculation in the nursery of ectomycorrhizal fungi in some eucalyptus genotypes cuttings can replace part of the phosphate fertilization in the nursery without compromising plant growth after planting in the field and still bring benefits in the face of some environmental stresses. Although, variations in response to inoculation are generally caused by previously mentioned factors, as well as the host-fungus compatibility, fungus effectiveness under local conditions, efficiency of the native fungi community [24], and the Eucalyptus genotype [12].

Field experiments under different conditions are necessary to optimize the benefits of inoculation; however, such reports are rare, and most studies found in the literature were carried out in the northern hemisphere addressing conifers. Thus, the objective of this study was to evaluate the field growth and colonization of Eucalyptus clones rooted cuttings previously EMF-inoculated in the production phase.

2. Material and Methods

2.1. Study Area and Soil Tillage

The experiment was carried out between January and December in the field of the Cabana Santa Bárbara farm of Gerdau Aços Longos S/A, in the municipality of Três Marias, Minas Gerais, Brazil (18°07′ S and 44°57′ W). The mean annual temperature of the area was 22.5 °C, the mean annual rainfall was 1442 mm [25], and the climate is classified as Aw-tropical climate with dry winters [26] (Figure 1).

The soil of the area used for the study was a Quartzipsamments soil [27] previously used for Eucalyptus cultivation, where leaf-cutting ants in the planting area and surroundings were contained 60 days before planting, in the week of planting (to eliminate hidden and undetected anthills in the first control) and by monitoring throughout the study period to prevent the establishment of new anthills. Soil tillage consisted only of opening plant rows.

2.2. Experimental Setup

To evaluate the effect of ectomycorrhizal inoculation on the growth of Eucalyptus trees after planting in the field, the treatments were applied in the rooted cuttings production phase and arranged as a 2 × 5 factorial in this study. The treatments were rooted cuttings of the Eucalyptus clones GG100 and GG680 previously inoculated with Pisolithus microcarpus isolate UFSC-Pt116, Hysterangium gardneri isolate UFSC-Hg93, or Scleroderma areolatum isolate UFSC-Sc129 plus two uninoculated controls were analyzed. The inoculated treatments and one uninoculated control (low P control) were produced on a substrate with reduced phosphate fertilization and the other control was produced on a substrate with full phosphate fertilization (high P control). In the nursery, the rooted cuttings were grown for 120 days as described by [12]. These rooted cuttings were then planted in a field experiment arranged in a randomized complete block design, with four replications. The experimental plots consisted of three rows with nine plants, totaling 27 plants per plot and 1080 in the entire experiment. Where, in each plot, the seven central plants were considered for evaluation, so, 280 trees were evaluated in the entire experiment.

2.3. Production of the Inoculant, the Substrate for Rooted Cutting, the Substrate Inoculation, and the Rooted Cuttings

All proceeding to obtain the rooted cuttings previously inoculated or not was the same described in [12], and the total phosphate fertilization, substrate plus fertigations, in the inoculated and low P control treatments provided a total of 4.2 mg P plant⁻¹, while in the high P control treatment the total phosphate fertilization was 34.3 mg P plant⁻¹. After 120 days, 10 rooted cuttings per treatment were randomly collected and the percentage of colonized root tips was evaluated [28] (

Table 1. Mean percentage of colonized root tips of 120 days-old rooted cutting of Eucalyptus clones GG100 and GG680 inoculated with Hysterangium gardneri, Pisolithus microcarpus, Scleroderma areolatum, and uninoculated controls (low P control and high P control).

Fungal Treatments	GG100	GG680
	--------------------------------------------- % ------------------------------------------------
Low P control	3.6 ± 1.37 1	3.2 ± 0.19
H. gardneri isolate UFSC-Hg93	5.3 ± 1.09	6.2 ± 1.06
P. microcarpus isolate UFSC-Pt116	4.1 ± 0.41	2.1 ± 1.42
S. aareolatum isolate UFSC-Sc129	5.9 ± 1.08	2.6 ± 0.67
High P control	1.0 ± 0.38	0.8 ± 0.28

¹ Standard deviation.

).

2.4. Fertilizers and Planting

Representative soil samples were collected from the 0–20 cm layer in and in-between the tree rows and sent to the Soil Fertility Laboratory of the UFVJM—the Federal University of the Jequitinhonha and Mucuri Valleys for physical and chemical characterization (

Table 2. Physical and chemical characterization of the soil at the planting site of Eucalyptus rooted cuttings.

Characteristics	Samples
	Into the Tree Rows	In-Between the Tree Rows
pH 1	4.6	4.4
P, mg dm−3	1.4	1.2
K, mg dm−3	9.3	6.2
Ca, cmolc dm−3	0.4	0.4
Mg, cmolc dm−3	0.2	0.2
Al, cmolc dm−3	0.82	0.88
H + Al, cmolc dm−3	6.5	6.5
SB, cmolc dm−3	0.62	0.62
t, cmolc dm−3	1.44	1.50
T, cmolc dm−3	7.12	7.12
m, %	57.0	59.0
V, %	9.0	9.0
M.O., dag kg−1	0.8	0.9
B, mg dm−3	2.0	2.0
Cu, mg dm−3	0.55	0.55
Fe, mg dm−3	68.7	65.9
Mn, mg dm−3	4.2	5.0
Zn, mg dm−3	2.45	2.90
Sand, %	79.0	79.0
Silt, %	2.0	2.0
Clay, %	19.0	19.0

¹ pH (H₂O) ratio 1:2.5 (soil:water); P and K: Mehlich-1; exchangeable Ca, Mg and Al: KCl 1 mol L⁻¹; H + Al: 0.5 mol L⁻¹ calcium acetate at pH 7.0; SB: base sum; t: effective cation exchange capacity (CTC); T: CTC pH 7.0; m: aluminum saturation; V: base saturation; M.O.: organic matter.

). Fertilization at planting was defined based on soil analysis data and applied according to the routine practices of the company. Then, 10 days before planting, 300 kg ha⁻¹ of NPK 3-26-5 (3% N, 26% P₂O₅; 5% K₂O) was applied to the 25–30 cm layer simultaneously with the furrows opening.

The 120-day-old rooted cuttings were planted by hand in 3 m × 3 m spacing; around the entire experimental area. To minimize the border effect, two rows of uninoculated and routinely fertilized rooted cuttings were planted.

Three months after planting, the plants were fertilized with 150 kg NPK ha⁻¹ (20% N, 0% P₂O₅; 20% K₂O) with 1% B. After five months, an amount of 900 kg ha⁻¹ dolomitic limestone and 300 kg ha⁻¹ gypsum was applied and after 11 months, 200 kg ha⁻¹ potassium chloride with 1% B.

2.5. Evaluations

The plant height, collar diameter, and chlorophyll levels were evaluated after 2, 6, and 12 months. The chlorophyll levels were determined indirectly by the chlorophyll index evaluated in the middle third of the first fully expanded leaf, from the plant apex to the base, and exposed to solar radiation with a ClorofiLOG^® chlorophyll meter model CFL 1030, and the results were expressed as Falker Chlorophyll Index (ICF). Survival was assessed after four months.

In the 6th and 12th months, diagnostic leaves were sampled to determine the nutrient concentration and fine roots were sampled to quantify the percentage of colonized root tips. The nutrient concentration was measured by the methodology described by [29]. The root samples were stored in 50% alcohol and the percentage of colonized root tips was determined by [28].

2.6. Statistical Analyses

The data distribution of plant height, collar diameter, the rooted cuttings survival rate in the field, chlorophyll levels, percentage of colonized root tips, and nutrient concentrations was analyzed by the Lilliefors test and homogeneity of variance by the Cochran and Bartlett test. The data were then subjected to analysis of variance and, when significant, the means were compared by the Tukey test at 5% probability.

3. Results

The plant survival rate in the field was not influenced by the type of clone or inoculation (

Table 3. Rooted cutting survival at four months after field planting, of the clones GG100 and GG680 of Eucalyptus, inoculated with Hysterangium gardneri isolate UFSC-Hg93, Pisolithus microcarpus isolate UFSC-Pt116, Scleroderma areolatum isolate UFSC-Sc129 and uninoculated controls (low P control and high P control).

Fungal Treatments	GG100	GG680	Means
	--------------------------------------------------------- % ---------------------------------------------------------
Low P control	100.0 ± 0.00 1	96.5 ± 7.00	98.3
H. gardneri	96.4 ± 7.14	96.4 ± 7.14	96.4
P. microcarpus	100.0 ± 0.00	100.0 ± 0.00	100.0
S. areolatum	100.0± 0.00	92.9 ± 8.25	96.4
High P control	100.0 ± 0.00	100.0± 0.00	100.0
Means	99.3	97.2	98.2

¹ Standard deviation.

).

After two months of planting, the mean plants height of both Eucalyptus clones inoculated with P. microcarpus was equal to those of the high P control treatment and taller than that of plants inoculated with other EMF and those of low P control treatment. Compared to low P control, the height of inoculated plants with P. microcarpus increased by 16% for clone GG100 and 13% for clone GG680 (

Table 4. Plant height and collar diameter at 2, 6, and 12 months after field planting, of plants of the clones GG100 and GG680 of Eucalyptus, inoculated with Hysterangium gardneri isolate UFSC-Hg93, Pisolithus microcarpus isolate UFSC-Pt116, Scleroderma areolatum isolate UFSC-Sc129 and uninoculated controls (low P control and high P control).

Fungal Treatments	2 Months			6 Months			12 Months
	GG100	GG680	Means	GG100	GG680	Means	GG100	GG680	Means
	--------------------------------------------------------------- Height, m ---------------------------------------------------------------
Low P control	0.37 ± 0.04 1	0.30 ± 0.05	0.33b	1.17 ± 0.30	1.32 ± 0.62	1.25	3.46 ± 0.52	3.36 ± 1.01	3.41
H. gardneri	0.40 ± 0.04	0.32 ± 0.05	0.36b	1.62 ± 0.27	1.47 ± 0.67	1.55	3.99 ± 0.41	3.65 ± 0.94	3.82
P. microcarpus	0.43 ± 0.06	0.34 ± 0.06	0.39a	1.65 ± 0.43	1.48 ± 0.83	1.56	3.70± 0.57	3.50 ± 1.15	3.6
S. areolatum	0.42 ± 0.07	0.32 ± 0.08	0.37b	1.43 ± 0.38	1.55 ± 0.95	1.49	3.69 ± 0.63	3.52 ± 1.58	3.61
High P control	0.52 ± 0.05	0.39 ± 0.05	0.45a	1.98 ± 0.07	1.62 ± 0.63	1.8	4.40 ± 0.38	3.53 ± 1.00	3.97
Means	0.43A2	0.33B	0.38	1.57	1.49	153	3.85	3.51	3.68
	-------------------------------------------------------- Collar diameter, cm --------------------------------------------------------
Low P control	0.37 ± 0.07	0.40 ± 0.05	0.39b	2.19 ± 0.34	2.03 ± 0.89	2.11	4.37 ± 0.53	4.42 ± 1.02	4.4
H. gardneri	0.52 ± 0.08	0.43 ± 0.11	0.47ab	2.99 ± 0.59	2.24 ± 0.85	2.61	5.18 ± 0.33	4.47 ± 1.06	4.94
P. microcarpus	0.53 ± 0.13	0.43 ± 0.15	0.48ab	2.86 ± 0.82	2.27 ± 1.05	2.57	4.63 ± 0.67	4.50 ± 1.13	4.56
S. areolatum	0.48 ± 0.17	0.43 ± 0.14	0.45b	2.34 ± 0.10	2.42 ± 1.44	2.38	4.63 ± 0.76	4.60 ± 1.71	4.62
High P control	0.73 ± 0.12	0.53 ± 0.11	0.63a	3.10 ± 0.15	2.51 ± 0.69	2.8	5.74 ± 0.40	4.72 ± 1.12	5.23
Means	0.53ª	0.44B	0.48	2.7	2.29	2.49	4.91	4.59	4.75

¹ Standard deviation; ² Means followed by the different letters, lowercase in columns and uppercase in rows, differ amongst themselves (p ≥ 0.05) by Tukey’s test.

).

Regarding the collar diameter, although no effect of the clones was observed and although the mean of the inoculated plants was equal to the low P control plants, two months after field planting the mean diameter of the inoculated clone GG100 plants was 38% higher than low P control plants (Table 4). This benefit in clone GG100 was even greater in plants inoculated with P. microcarpus, as the diameter was 43% larger than in low P control plants. For clone GG680, the trend was the same, but the increase was smaller (8%). Only two months after planting, the mean collar diameter of the plants inoculated with H. gardneri and P. microcarpus was equal to that of the high P control plants. No differences in plant height and collar diameter were observed in the evaluations at 6 and 12 months after planting (Table 4).

Chlorophyll levels were not influenced by EMF-inoculation, and these levels were highest in the clone GG100 plants, in all evaluations (data not shown). After 6 and 12 months, the leaf’s nutrient concentrations of the rooted cuttings were not influenced by inoculation or phosphate fertilization reduction in the nursery stage.

Ectomycorrhizal colonization occurred in both inoculated and uninoculated plants and was similar among clones. In the percentage of colonized root tips (mean of 3.5% at planting, Table 1), an increase was observed after six months (mean of 7.3%) as observed in

Table 5. Percentage of colonized root tips at two, six, and 12 months after field planting, of plants of the clones GG100 and GG680 of Eucalyptus, inoculated with Hysterangium gardneri isolate UFSC-Hg93, Pisolithus microcarpus isolate UFSC-Pt116, Scleroderma areolatum isolate UFSC-Sc129 and uninoculated controls (low P control and high P control).

Fungal Treatments	GG100	GG680	Means	GG100	GG680	Means
	----------------- 6 Months ----------------			---------------- 12 Months -----------------
	------------------------------------------------------ % -------------------------------------------------------
Low P control	7.1 ± 2.26 1	6.7 ± 1.46	6.6bc 2	36.2 ± 16.17	31.4 ± 7.57	33.8
H. gardneri	3.4 ± 0.57	4.4 ± 0.95	3.9c	30.5 ± 10.94	31.6 ± 6.58	31.1
P. microcarpus	6.9 ± 0.78	8.9 ± 1.55	7.9b	31.8 ± 4.65	29.2 ± 6.19	30.5
S. areolatum	13.6 ± 3.32	14.3 ± 6.01	13.9a	33.9 ± 11.33	29.9 ± 4.17	31.9
High P control	4.8 ± 1.71	3.3 ± 0.34	4.1c	31.8 ± 6.29	32.5 ± 7.35	32.1

¹ Standard deviation; ² Means followed by the different letters in columns differ amongst themselves (p ≥ 0.05) by Tukey’s test.

. After six months, the mean percentage of colonized root tips of both clones was higher in the plants inoculated with S. areolatum, followed by those inoculated with P. microcarpus and of the low P control. The percentages of colonized root tips were lowest in the plants inoculated with H. gardineri and the high P control plants (Table 5). After 12 months, the percentage of colonized root tips was similar in all treatments and in the mean 4.3 times higher than the percentage observed after 6 months.

4. Discussion

The survival rate observed in this study was high and the absence of an effect of inoculation under commercial cultivation conditions and without stress (Table 3) occurrence confirms the results observed for conifers [19,22]. However, although not significant, the 4% higher survival rate for both clones, of high P control and P. microcarpus treatments than those of low P control treatment can represent financial gains. Considering that Eucalyptus spp. is one of the most widely planted broad-leaf forest species in the world. As many as 95 countries have eucalyptus planted in plantations, and the total plantation area has exceeded 22.57 million hectares worldwide [30] and that 10% of this area is replanted every year with approximately 1,333 rooted cuttings (or seedlings) per hectare, the world total annual demand is approximately 30 million rooted cuttings. Thus, a 4% higher survival rate could represent an economy of 1.2 billion rooted cuttings. It is important to note that this 4% increase in survival was observed in a crop that did not experience stress conditions, such as drought, nutritional deficiency, or pathogen attack, that usually happens. Since the rooted cutting was planted in January when the rainfall was up to 200 mm, no pathogen attack was registered, and routine fertilizers developed for uninoculated root cuttings were used. Under stress conditions, the benefits could be greater. As is general knowledge, the greatest benefits of ectomycorrhizae are observed under some types of stress such as drought, pathogens, or heavy metal excess [31,32,33]. Thus, inoculation may contribute to a cost reduction in the establishment of eucalyptus plantations sparing the replanting stage, and consequently, the formation of stands without failures, which can contribute to greater productivity of the area.

Increasement of growth was observed for height and diameter of GG100 clone, mainly the plants inoculated with P. microcarpus isolate UFSC-Pt116, compared with the low P control plants after two months (Table 4), indicating that inoculated plants benefit from ectomycorrhiza formation, contributing to their development. The beneficial effect on the survival, height, and diameter for the inoculation of the P. microcarpus isolate UFSC-Pt116 observed after transplanting the seedlings to the field (Table 3, Table 4 and Table 5) confirms its ability to increase the growth of Eucalyptus plants previously observed in the nursery stage [12,15,16,17]. In a small greenhouse experiment, seedlings of E. dunnii, this isolate (UFSC-Pt116), and other two fungi promoted the production of greater shoot dry matter, compared to treatments inoculated with the other fungus tested, and to the uninoculated controls without P fertilization and low P fertilization (NI 0 P and NI 0.5 P) and, produced as much biomass as the uninoculated controls fertilized with the highest dosage of P (NI 8 P) (12). In a larger experiment (70 plants per experimental plot with four replications) conducted under a commercial nursery and without substrate sterilization, the same isolate (UFSC-Pt116) promoted a higher frequency of a firm root ball and well-rooted system, greater height, larger diameter, and greater shoot dry mass and total dry mass observed in the rooted cuttings clones of E. urophylla and E. grandis hybrids compared with the low P control uninoculated and the other inoculated cuttings [12]. It is noteworthy that in this cited study, the inoculated rooted cuttings grew as much as the non-inoculated ones, which were produced with approximately 8.2 times more phosphate fertilization.

These benefits obtained from the ectomycorrhizal association are an alternative to the use of fertilizers based on polluting or non-renewable sources. However, these results demonstrate the importance of EMF selection and indicate the need to evaluate whether Eucalyptus clones respond differently to ectomycorrhizal inoculation, as observed in nursery conditions by [12]. In a study with two Eucalyptus clones, although both clones had their rooted cuttings shoot height and root collar diameter improved by Pisolithus microcarpus isolate UFSC-Pt116 inoculation, only the clone GG680, an intraspecific hybrid between E. urophylla × Eucalyptus grandis, also have the total dry mass and shoot dry mass of the rooted cuttings higher than low P control and other inoculated cuttings [12]. If certain Eucalyptus clones are more responsive than others, this characteristic may have to be integrated into breeding programs, with a view to the selection of plants that allow the establishment of more sustainable forest tree plantations stands, less dependent on phosphate fertilization.

The effects of previous EMF-inoculated rooted cuttings of Eucalyptus plants on the field, observed only at the two months evaluation (Table 4), could be related to low colonization percentage in the nursery (Table 1), isolates low adaptation concerning pre-existing fungi in the area, or the natural colonization of uninoculated rooted cuttings after planting in the field by pre-existing fungi (Table 5) since the study area was previously used for Eucalyptus cultivation and is surrounded by Eucalyptus plantations. The evaluated isolates were obtained from southern Brazil (Santa Catarina) and had not been tested until then in the environmental conditions of the southeast of Brazil (Minas Gerais). This result indicates the need for evaluations of a greater number of isolates from different regions of origin. If the influence of the regional origin on the efficiency of ectomycorrhizal isolates is confirmed, environment-specific isolates with greater capacity to adapt to different environments need to be selected.

Although an intensive growth of the plants previously inoculated in the nursery compared to the low P control was observed, only at the two months evaluation (Table 4), this result is expressive since they were obtained in a plantation free of environmental and nutritional stresses, which are very common under field conditions for large, planted areas, caused for example by dry summer spells or even prolonged drought, when planting is carried out at suboptimal conditions. It is worth remembering that the seedlings were fertilized with top- and side-dressing according to the optimal recommendations established over many years of research. Thus, these results emphasize the high biotechnological potential of EMF, mainly considering that most soils in the world have some nutrient deficiency [13] and that it may be impossible to maintain the current levels of fertilization, particularly the phosphatic and nitrogenic fertilization, given the expectation of natural resource scarcity and soaring costs. Thus, new studies should be developed with more responsive clones, more promising isolates, and varying levels of mainly phosphate fertilizers.

The colonization observed in this study (Table 5) is lower than that reported after six months and similar to that after a year in Eucalyptus globulus pre-inoculated with EMF [34]. The plants’ EMF-colonization at six months was lower than that observed in established plantations of Eucalyptus grandis without previous inoculation, but was higher than colonization after 12 months, in an area where the management included burning, and similar to an area with minimum tillage [35]. During the field evaluation, the percentage of colonized root tips may have increased due to colonization by native fungi, since the rooted cuttings taken to the field had non-colonized sites (Table 1). The fungi inoculated in the nursery may have been replaced by native ones, promoting a colonization increase over the following months in the field [22,36]. The low survival of the fungus inoculated at some sites may partially explain why inoculation with effective ectomycorrhizal isolates does not always increase plant growth in the field [37]. The increase in colonization in the field period (Table 5) may also have been favored by the lower availability of P in the soil than in the conditions under which the rooted cuttings were produced.

The similar levels of nutrients for plants of all treatments can be explained by the same top- and side-dressing fertilization for all plants.

Field experiments may be affected by the variation in climatic, edaphic, and microbial factors that cannot be controlled, and which tend to influence and often confuse responses to EMF-inoculation treatments [20]. Therefore, other studies under similar conditions should be performed to describe the EMF action and their contribution to plant growth. Moreover, molecular techniques must be used to identify the inoculated fungi, so that later it is possible to verify whether the colonizing fungus is the same as the one initially inoculated, and thus select fungi based on their effectiveness and colonization efficiency, and promote benefits after planting in the field. The results demonstrate the need for new experiments with different fungi and isolates from different origins that can provide a higher percentage of colonized root tips in the nursery stage and the use of lower doses of fertilizers, especially phosphates.

5. Conclusions

Inoculation with ectomycorrhizal fungi (EMF) in the tree nursery boosts the initial plant growth in the field, but this effect depends on the type of ectomycorrhizal fungus, clone, and fertilization used in the nursery. Ectomycorrhizal colonization of Eucalyptus plants occurs naturally and increases with the development of the trees in the field. The low phosphate fertilization in the production of EMF-inoculated seedlings did not influence the plant development after field planting.