Influence of Mycorrhizal Fungi on the Rooting of Stem and Stolon Cuttings of the Colombian Blueberry (Vaccinium meridionale Swartz)

Abstract

The Colombian blueberry (Vaccinium meridionale Swartz) is a promissory crop for the Andean Region of South America, and the absence of a suitable propagation protocol presents a difficulty introducing it into commercial production. This research was undertaken in order to evaluate the effect of mycorrhizal fungi on rhizogenesis of stem and stolon cuttings of the Colombian blueberry. The highest rooting percentages (up to 73.3%) were observed at day 60 on stem cuttings treated with 200 mg ⁻¹ 4-indole-3yl-butyric acid or napthtalene-acetic acid in mycorrhiza-free substrate. The presence of mycorrhizas in substrate increased cutting viability up to 95%, but reduced percentage rooting, root number, and root length on stem cuttings of the Colombian blueberry. Treating stem cuttings with 200–400 mg ⁻¹ 4-indole-3yl-butyric acid or napthtalene-acetic acid diminished the effects of fungi on root formation. The use of stolon cuttings for vegetative propagation of the Colombian blueberry had low practical applicability. The results of the study might further serve to establish the optimal substrate conditions and cutting source for vegetative propagation of the Colombian blueberry in commercial culture.

KEYWORDS:

• Vaccinium meridionale
• mycorrhiza
• propagation
• adventitious roots

INTRODUCTION

The Colombian blueberry (Vaccinium meridionale Swartz, Ericaceae), known also as agraz, Andean blueberry, or Jamaican bilberry, is a promissory crop growing in the mountainous areas of Colombia, Peru, Ecuador, Venezuela, and Jamaica. It is a wild ramified evergreen shrub of 0.2–5-m height reaching 1–2-m height during the period of maximum fruit production. From the approximately 35 species of genus Vaccinium found in South America (Salinas and Betancur, 2007), only the Colombian blueberry and ‘mortiño’ (V. floribundum Kunth.) possess edible fruits (Schick, 1993). The fruits of the Colombian blueberry are rich in anthocyanins, especially delphinidin (Ramírez et al., 2007), sugars, and vitamin C (Ávila et al., 2007) and are traditionally used in the Northern Andean region of South America to prepare marmalades, vines, torts, or for fresh consumption or textile coloration; the plant is also utilized for ornamental purposes. The local practices for propagation of the Colombian blueberry are essentially artisanal and unsophisticated. In Colombia and adjacent countries, there is an increasing demand in a protocol for propagation of the Colombian blueberry that should be suitable, cheap, and accessible for small growers.

Vegetative propagation by stem cuttings is traditionally used to multiply various Vaccinium sp. plants (Fischer et al., 2008; Litwinczuk et al., 2005; Morrison et al., 2000) and may be equally employed for the Colombian blueberry (Castrillón et al., 2008); the disadvantages of this method could include slow rates of rhizogenesis and low rooting percentage. Other propagation techniques, such as by seeds, have different shortcomings, including slow germination rate, seed dormancy, and low uniformity of seedling emergence (Magnitskiy and Ligarreto, 2007). Micropropagation of Vaccinium sp. has distinct advantages over propagation by cuttings in terms of rooting percentage and plantlets survival (Meiners et al., 2007; Morrison et al., 2000), but it is not currently available to the growers of Andean region. For vegetative propagation of Vaccinium sp., rooting hormones are commonly applied to increase rooting percentage in stem cuttings, whereas the efficiency of the hormone application is highly variable depending on plant species, health, and growth stage of mother stock plant (Miller et al., 2006); treatment conditions (Lee et al., 2004); and use of hardwood (Fischer et al., 2008), or softwood (Castrillón et al., 2008; Morrison et al., 2000) cuttings.

Much less information is available on the potential use of stolon or rhizome cuttings for propagation of Vaccinium sp. At the same time, the rooting of stolons and rhizomes constitutes a common way of propagation of the Colombian blueberry and other shrubs and creeping herbs under natural conditions of a tropical forest. Rooting of rhizomes and stolons represents a competitive strategy for rapid establishment of Vaccinium sp. plants under conditions of reforestation (Moola and Mallik, 1998) and, while the enhanced rhizome formation may not necessarily contribute to increased fruit production (Jamieson and Nickerson, 2003), it explains the pioneering role of the Colombian blueberry during revegetation of a tropical mountain forest (McDonald et al., 2003). The use of stolon cuttings, if technically sustained, might serve as an additional method for vegetative propagation of the Colombian blueberry in commercial nurseries.

A factor that apparently influences growth of the Colombian blueberry is the association of this plant with mycorrhizal fungi. Under natural conditions of the Colombian highlands, the Colombian blueberry is frequently found as an understory shrub in oak forests of low light intensity, soils low in nitrogen, and characterized by pH 5.0–5.5 and high presence of mycorrhizas. Mycorrhizas of the Ericaceae are known to improve plant nutrition with P, N, K, S, and Cu (Smith and Read, 2008; Yang et al., 2002) and to increase stress tolerance in plants (Cairney and Meharg, 2003). However, there is a lack of published data on the importance of mycorrhizal associations for the growth and propagation of the Colombian blueberry. In propagation practice, the rhizogenesis of unrooted cuttings may be affected by the presence or absence of mycorrhizal fungi in the rooting media (Davies, 2000). The capacity of the Colombian blueberry cuttings to form adventitious roots in mycorrhiza-enriched substrates has not been evaluated, whereas the positive (Davies, 2000; Powell and Bagyaraj, 1984; Scagel et al., 2005) as well as the negative (Lanthier, 2006; Repac, 1998) effects from the presence of mycorrhizas in substrate might be expected.

Previous studies suggested the potential benefits of treating stem cuttings of the Colombian blueberry with 200 or 400 mg L⁻¹ 4-indole-3yl-butyric acid (IBA) or napthtalene-acetic acid (NAA) to increase rooting in organo-mineral substrates (Castrillón et al., 2008). The purpose of the present study was to evaluate the effect of mycorrhizal fungi, when present in the substrate, on rhizogenesis of stem and stolon cuttings of the Colombian blueberry. The capacity of the Colombian blueberry cuttings to form adventitious roots in mycorrhiza-enriched substrate under the presence of rooting hormones was also studied.

MATERIALS AND METHODS

Plant Material and Substrates

The entire adult 4–5-year-old year shrubs of the Colombian blueberry (Vaccinium meridionale Swartz) were collected in the oak-pine forest in the municipality of Guachetá, Colombia (5.23° N, 73.41° W, 2,688 m over sea level, 14°C average annual temperature) and transported to the Faculty of Agronomy, National University of Colombia, Bogotá campus. The experiments were conducted in the plastic-covered unheated greenhouses (4.36° N, 74.40° W, 2,640 m over sea level, 18°C average day temperature, 7°C average night temperature), where propagation material (stem and stolon cuttings) taken from homogenous plants with good vigor and without visible nutritional or pathological disorders was used.

Stem softwood cuttings (4-cm average length) having four to five green leaves and the apical bud, and stolon tip cuttings (8-cm average length) consisting of five to six phytomers and the apical bud were used. Immediately after collection, the basal ends of each cutting (approximately 1 cm) were moistened with distilled water and dipped into a rooting paste containing technical talc and 0, 200, or 400 mg L⁻¹ chemically pure grade IBA or NAA according to Castrillón et al. (2008). This protocol allowed using lower concentrations of hormones for successful rooting of Vaccinium sp. rather than immersing cutting bases in highly concentrated solutions of hormones (Fischer et al., 2008). Excess paste was tapped off after 20 min drying, the cuttings were set into 3,000 cm³ plastic containers filled with 1:1 v/v mixtures of peat:coal ash (mycorrhiza-free substrate) or mycorrhiza:coal ash (mycorrhiza-enriched substrate) and arranged on a greenhouse bench under an intermittent mist. The peat employed to prepare a mycorrhiza-free substrate was Base Substrate Klasmann® (Klasmann-Deilmann, Geeste, Germany). The peat-based commercial product used for inoculation with mycorrhizas was Micorrizafer® (Agrosafer Company, Medellín, Colombia) containing mycorrhiza propagules (spores, mycelium, and old colonized roots) of a mixture of four mycorrhizal genera (Glomus, Entrophospora, Scutellospora, and Acaulospora) with minimal concentration of 230 spores per gram of substrate. The pH was 5.5 in both substrates. The stem cuttings were inserted vertically in the rooting media, while stolon cuttings were placed horizontally on the surface of the substrates. Relative humidity in the greenhouse was maintained between 90% and 95%. Plastic black mesh was additionally established over the top of the greenhouse bench to provide 50% shading.

Treatment plots were arranged in a completely randomized design over the greenhouse bench. Each treatment plot consisted of a row of five stem cuttings or four stolon cuttings per ten treatments, and each treatment was replicated four times per trial, making 200 or 160 cuttings of stems or stolons, respectively. After a 60-day growth period, the cuttings were removed from containers, and the roots were carefully washed to remove all substrate particles. The number of viable cuttings, number of roots, and root length were measured and rooting percentage per number of viable cuttings was calculated for each treatment.

Colonization of the Roots with Mycorrhizal Fungi

When sampling roots to measure the amount of mycorrhizal colonization, fine fibrous roots were selected from all stem or stolon cuttings at day 60, and the substrate media were washed gently from the roots. The roots were cleared using hot 10% KOH in a water bath at 90°C during 20 min, washed thoroughly with distilled water three times, and immersed in 10% HCl for 15 min at 90°C following the protocol described by Koske and Gemma (1980). The roots were then placed for 20 min in 0.05% trypan blue (5 trypan blue:95 lactic acid v/v) at 90°C. The stain was prepared by mixing with distilled water, glycerin, and lactic acid in proportions of 1:1:1 v/v/v. This step was done without application of heat, but the incubation period was extended to 12 h. The stain was poured off into another container, and the roots were placed in water-glycerin mix (50%). Following this treatment, the percentage of the root length colonized by mycorrhizal fungi was detected using the gridline intersect technique (Giovannetti and Mosse, 1980).

Statistical Analysis

Analysis of variance (ANOVA) procedures were used to test for significant effects of the cutting and stolon traits for the variables studied (SAS Institute, Inc., 1996). A probability level of 0.05 was judged to be significant in all statistical tests. Significant differences between means for each group of cuttings were estimated using the Duncan Multiple Range Test, p = 5%. The closeness of relationship among selected variables was determined by correlation analyses using the Pearson linear correlation coefficients (SAS Institute, Inc., 1996).

RESULTS AND DISCUSSION

Stem Cuttings

Stem cuttings treated with 200 or 400 mg L⁻¹ IBA and placed in a substrate with no mycorrhiza added had an increment in viability compared to respective water-treated controls, although high viability rates were not necessarily associated with high rooting percentage at day 60 (Table 1). When growing in mycorrhiza-free substrate, cuttings had a higher rooting percentage, up to 68.8% or 73.3% when treated with paste containing 200 mg L⁻¹ IBA or NAA, respectively, whereas rooting in the control treatment was 60.2% (Table 1). Treating a cutting base with either 200 mg L⁻¹ IBA or NAA in mycorrhiza-free substrate significantly increased root number and root length as compared with controls (Table 1). The usage of 400 mg L⁻¹ IBA or NAA did not contribute to higher average number of roots or root length (Table 1) in accordance with the previous data, suggesting that the application of IBA or NAA in rates that exceeded 200 mg L⁻¹ do not increase rooting in stem cuttings of the Colombian blueberry (Castrillón et al., 2008).

TABLE 1 Effect of Mycorrhiza Presence in Substrate on the Rooting of Stem Cuttings of the Colombian Bblueberry (Vaccinium meridionale Swartz) Treated with Rooting Hormones

		Viable cuttings (%)		Rooting (% viable cuttings)		Average number ofroots per cutting		Average root length (cm)
		−Mycor	+Mycor	−Mycor	+Mycor	−Mycor	+Mycor	−Mycor	+Mycor
H2O (control)		71 ab	95 a	60.2 ab	21.0 b	1.8 c	0.2 b	3.15 bc	0.72 c
IBA	200 mg L^−1	80 a	64 b	68.8 a	38.5 a	6.0 a	1.8 a	8.15 a	3.30 a
	400 mg L^−1	90 a	60 b	50.0 ab	41.7 a	3.2 b	0.8 ab	2.23 c	0.75 c
NAA	200 mg L^−1	75 ab	40 c	73.3 a	50.0 a	4.8 ab	2.2 a	6.95 a	2.15 ab
	400 mg L^−1	55 b	65 b	36.4 b	46.2 a	2.0 c	3.3 a	2.15 c	1.43 b
Treatment means in the same column followed by the same letter are not significantly different at 5% level of Duncan Multiple Range Test.

When plants were propagated in mycorrhiza-enriched substrate, viability of cuttings in control treatments (no hormone applied) was higher (95%) than that in mycorrhiza-free substrate (Table 1). At the same time, rooting percentage was reduced in control cuttings placed in mycorrhiza-enriched substrate (21%) as compared with those growing in mycorrhiza-free substrate (60.2%).

These results indicated that the presence of mycorrhiza fungi in a substrate was beneficial for maintenance of cutting viability, whereas percentage rooting, average number of roots, and root length were reduced. It is well known that mycorrhizas can improve viability of unrooted cuttings (McLean et al., 1994; Scagel, 2001; Scagel et al., 2005), such as via fungi-derived hormonal substances (Barea and Azcon-Aguilar, 1982), while the influence of mycorrhizal fungi on rhizogenesis of cuttings is not always positive (Lanthier, 2006; Repac, 1998). It may be speculated that, in our experiment, the allocation of plant assimilates to the hyphal growth competed with the allocation of carbohydrates to the secondary root formation (Johnson et al., 1997; Smith and Read, 2008), and as a result, a negative effect of mycorrhizal fungi on the rhizogenesis was observed. The allocation of plant assimilates to the fungi during the establishment of a mycorrhizal association is well studied (Jakobsen and Rosendahl, 1990), while a similar process occurs during the establishment of a less mutual relationship for the plant (Johnson et al., 1997; Smith and Read, 2008). In the latter case, especially under low levels of available nutrients in a substrate, mycorrhizal fungi might become parasitic (Johnson et al., 1997) and maintain viability of cuttings, while exploring plant-derived metabolites and, thus, limiting formation of adventitious roots. It may be argued, however, that the inhibition of root formation attributed to the presence of mycorrhizas in our experiment was short-termed and due to the lag-phase, a temporally inhibition of root growth at the initial stages of colonization (Smith and Read, 2008). Additionally, the absence of roots in cuttings would delay the formation of mycorrhizal association. However, while the Colombian blueberry is a plant recalcitrant to vegetative propagation, approximately 40 days are usually sufficient for the cuttings to initiate development of adventitious roots (Ligarreto and Magnitskiy, 2008 unpublished data). It is also worth noticing that a level of genotype-genotype specificity between the host and the fungus defines a range of responses in a mutualism-parasitism continuum, ranging from a response negative for the plant to a positive one, especially in the case of Vaccinium sp. plants, for which few commercial products containing specialized mycorrhizal fungi are available (Lanthier, 2006; Vega et al., 2009).

With hormones applied to the cutting base in mycorrhiza-enriched substrate, both cutting viability and rooting did not exceed 65% and 50%, respectively (Table 1). The presence of mycorrhiza had a more adverse effect on rooting for water-treated cuttings (21%) than for hormone-treated ones. When IBA or NAA were applied to the cutting base, the negative effect of mycorrhizas on rooting percentage was less noticeable, resulting in up to 41.7% or 50% rooting, respectively. These antagonistic effects of hormones and mycorrhizas on rhizogenesis in mycorrhiza-enriched substrate may be explained from the point of view that hormones, such as IBA or NAA, when present in substrate, had a negative affect on a fungus growth (Lanthier, 2006) or signaling between the fungi and the plant.

In summary, stem cuttings grown in mycorrhiza-enriched substrate had lower rooting percentage, reduced number of roots, and shorter roots compared to their counterparts grown in mycorrhiza-free substrate. Treating cuttings with 200 mg L⁻¹ IBA or NAA in mycorrhiza-enriched substrate significantly increased root number and root length compared to that of control cuttings. A similar trend was noticed for cuttings in mycorrhiza-free substrate. However, for most treatments, both root number and root length were lower in the presence of mycorrhizas compared to that in mycorrhiza-free substrate (Table 1).

Colonization of the root length by mycorrhizal fungi was equal to or exceeded 50% in both substrates (Table 2). Mycorrhizal colonization was, in general, lower in controls (water-treated) than in hormone-treated cuttings for both mycorrhiza-enriched and mycorrhiza-free substrates (Table 2). The high rates of root length colonization in substrate with no external mycorrhizas added (Table 2) may be explained by the initial presence of fungi in a nonsterilized substrate.

TABLE 2 Colonization of Roots of Stem Cuttings of the Colombian Blueberry (Vaccinium meridionale Swartz) Treated with Rooting Hormones with Mycorrhizal Fungi

		Colonization of the root length (%)
		−Mycor	+Mycor
H2O (control)		50.0 b	60.0 b
IBA	200 mg L^−1	78.6 a	69.2 ab
	400 mg L^−1	50.0 b	80.3 a
NAA	200 mg L^−1	57.1 ab	75.0 ab
	400 mg L^−1	60.0 ab	90.0 a
Treatment means in the same column followed by the same letter are not significantly different at 5% level of Duncan Multiple Range Test.

In the substrate with mycorrhiza added, strong correlations were found among cutting viability or rooting percentage, on the one side, and percentage of colonization, on the other side. For hormone-treated cuttings (0, 200, and 400 mg L⁻¹) in mycorrhiza-enriched substrate, the negative correlations among cutting viability and percentage colonization were found: −0.89 for IBA and −0.54 for NAA. The positive correlations among rooting percentage and percentage of colonization for the same treatments were 0.91 for IBA and 0.80 for NAA. In other words, increasing rates of hormones were associated with both high rooting percentage and high colonization of roots, if the latest were formed. However, in general, reduced root formation was observed in mycorrhiza-enriched substrate (Table 1) and this effect of fungi was less noticeable when increasing rates of IBA or NAA (Table 1).

Stolon Cuttings

The data on viability and rooting for stolon cuttings were much less consistent than that for stem cuttings (Table 3), which makes interpretation of the results difficult. In various treatments, the stolon cuttings were dried off by day 30 from the start of the experiment. The pattern in viability in mycorrhiza-enriched substrate at day 60 after planting was similar for both stolon and stem cuttings; more viable cuttings were registered when no hormones were applied (Tables 1, 3). In some treatments (200 mg L⁻¹ IBA, 400 mg L⁻¹ NAA), stolon cuttings grown in mycorrhiza-free substrate remained viable up to the end of the experiment but did not produce roots. When treated with IBA or NAA, the viability of stolon cuttings was, similarly to that observed in stem cuttings (Table 1), higher in mycorrhiza-free substrate and lower in mycorrhiza-enriched substrate compared to respective controls (Table 3). For some treatments with rooting hormones, such as 200 mg L⁻¹ IBA or NAA, all cuttings that remained viable in mycorrhiza-enriched substrate produced roots.

TABLE 3 Effect of Mycorrhiza Presence in Substrate on Rooting of Stolon Cuttings of the Colombian Blueberry (Vaccinium meridionale Swartz) Treated with Rooting Hormones

		Viable cutting (%)		Rooting (% viable cuttings)		Average number of roots per cutting		Average root length (cm)
		−Mycor	+Mycor	−Mycor	+Mycor	−Mycor	+Mycor	−Mycor	+Mycor
H2O (control)		0 c	40 a	0 c	50 b	0 b	2.7 b	0 c	0.40 ab
IBA	200 mg L^−1	20 b	30 ab	0 c	100 a	0 b	3.6 a	0 c	1.05 a
	400 mg L^−1	60 a	0 c	35 b	0 c	0.2 ab	0 c	0.30 a	0 c
NAA	200 mg L^−1	20 b	20 ab	100 a	100 a	0.2 ab	0.2 c	0.15 b	1.10 a
	400 mg L^−1	10 bc	10 c	0 c	0 c	0 b	0 c	0 c	0 c
Treatment means in the same column followed by the same letter are not significantly different at 5% level of Duncan Multiple Range Test.

For stolon cuttings, in both substrates, only control (water-treated) cuttings presented 60% colonization, whereas in all other treatments, colonization of root length did not exceed 5% with no significant differences observed among the treatments (data not shown). Thus, in contrast to the data obtained for stem cuttings, percentage colonization of root length was much lower in hormone-treated than in water-treated stolon cuttings.

Stolon cuttings may be successfully used for vegetative propagation of various species, including white clover (Annicchiarico and Piano, 2004; Murray et al., 2002) and strawberry (Smith, 1972), and application of rooting hormones to stolon cuttings might increase rhizogenesis in these. Thus, according to Hamner and Marth (1943), application of 50, 200, or 1,000 mg L⁻¹ IBA or NAA in a mixture with talc to the base of 4-cm long stolon cuttings of goldenrod (Solidago sp.) increased rooting percentage and contributed to production of vigorous adventitious roots. Treating the base of stolon cuttings of a medicinal herb Picrorhiza kurrooa (Royle ex Benth) with 100–200 mg L⁻¹ IAA or IBA increased percentage survival of cuttings and reduced the number of days required for rooting; the effect was proved on top segments of stolon cuttings but not on the basal segments (Nautiyal et al., 2001).

The plants of the genus Vaccinium present a long-term persistence in the understory of many second-growth forests under extremely low light conditions that favor storage of photosynthates in plagiotropic shoots and roots at the expense of above-ground stems and foliage (Alaback and Tappeiner, 1991; Moola and Mallik, 1998), suggesting the possible use of stolons or rhizomes for propagation practices. Low viability and poor rooting performance of stolon cuttings in our study might be due to the excessive light conditions, inadequate age (softwood) or length of cuttings, insufficient number of vegetative buds or bud dormancy, or inadequate hormone concentration in hormone-treated cuttings, among others. It may also be that fungal (Cline, 2004) or bacterial (Kobayashi et al., 1995) infections typical for Vaccinium sp., while not visually observed in our study, decreased viability of fresh stolon cuttings when these were placed in a contact with substrates.

According to our results, the use of vegetative stem cuttings is a relatively fast and simple method for propagation of the Colombian blueberry, despite the fact that Vaccinium sp. cuttings have low rates of adventitious root development (Litwinczuk et al., 2005; Miller et al., 2006). The presence of mycorrhizas and the use of a nonsterile substrate, in general, contributed to reduced root development on stem cuttings of the Colombian blueberry. While using hormones may diminish the effects of fungi on root development in cuttings of the Colombian blueberry, a sterilization of rooting media might be further suggested to increase the rates of rhizogenesis in unrooted cuttings. In future experiments, the benefits of inoculation of previously rooted stem cuttings with mycorrhizal fungi, as well as changing the nutrient levels in the rooting media or inoculation rates, may be addressed. The use of stolon cuttings for vegetative propagation of the Colombian blueberry has low practical applicability. The results of the study might further serve to establish the optimal substrate conditions and source of stem cuttings for vegetative propagation of the Colombian blueberry in commercial culture.

ACKNOWLEDGMENTS

This research was conducted under the project “Estandarización de métodos de propagación y escalamiento de producción de plántulas de agraz (Vaccinium meridionale Swartz)” funded by The Ministry of Agriculture and Rural Development of Colombia and The National University of Colombia, Bogotá, Colombia.