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Influence of Mycorrhizal Fungi on theRooting of Stem and Stolon Cuttings ofthe Colombian Blueberry (Vacciniummeridionale Swartz)Rodrigo A. Ávila Díaz-Granados a , Oscar J. Orozco Silva a , GustavoLigarreto Moreno b , Stanislav Magnitskiy b & Alia Rodríguez ba National University of Colombia , Bogotá, Colombiab Faculty of Agronomy , National University of Colombia , Bogotá,ColombiaPublished online: 09 Dec 2009.

To cite this article: Rodrigo A. Ávila Díaz-Granados , Oscar J. Orozco Silva , Gustavo LigarretoMoreno , Stanislav Magnitskiy & Alia Rodríguez (2009) Influence of Mycorrhizal Fungi on the Rooting ofStem and Stolon Cuttings of the Colombian Blueberry (Vaccinium meridionale Swartz), InternationalJournal of Fruit Science, 9:4, 372-384, DOI: 10.1080/15538360903378575

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International Journal of Fruit Science, 9:372–384, 2009Copyright © Taylor & Francis Group, LLC ISSN: 1553-8362 print/1553-8621 onlineDOI: 10.1080/15538360903378575

WSFR1553-83621553-8621International Journal of Fruit Science, Vol. 9, No. 4, Oct 2009: pp. 0–0International Journal of Fruit Science

Influence of Mycorrhizal Fungi on the Rooting of Stem and Stolon Cuttings of the Colombian

Blueberry (Vaccinium meridionale Swartz)

Mycorrhizas in Rooting of the Colombian BlueberryR. A. Ávila Díaz-Granados et al.

RODRIGO A. ÁVILA DÍAZ-GRANADOS1, OSCAR J. OROZCO SILVA1, GUSTAVO LIGARRETO MORENO2, STANISLAV MAGNITSKIY2,

and ALIA RODRÍGUEZ2

1National University of Colombia, Bogotá, Colombia2Faculty of Agronomy, National University of Colombia, Bogotá, Colombia

The Colombian blueberry (Vaccinium meridionale Swartz) is apromissory crop for the Andean Region of South America, and theabsence of a suitable propagation protocol presents a difficultyintroducing it into commercial production. This research wasundertaken in order to evaluate the effect of mycorrhizal fungi onrhizogenesis of stem and stolon cuttings of the Colombian blue-berry. The highest rooting percentages (up to 73.3%) were observed atday 60 on stem cuttings treated with 200 mg -1 4-indole-3yl-butyricacid or napthtalene-acetic acid in mycorrhiza-free substrate. Thepresence of mycorrhizas in substrate increased cutting viability upto 95%, but reduced percentage rooting, root number, and rootlength on stem cuttings of the Colombian blueberry. Treating stemcuttings with 200–400 mg -1 4-indole-3yl-butyric acid or napth-talene-acetic acid diminished the effects of fungi on root forma-tion. The use of stolon cuttings for vegetative propagation of theColombian blueberry had low practical applicability. The results ofthe study might further serve to establish the optimal substrateconditions and cutting source for vegetative propagation of theColombian blueberry in commercial culture.

KEYWORDS Vaccinium meridionale, mycorrhiza, propagation,adventitious roots

Address correspondence to Dr. Stanislav Magnitskiy, Faculty of Agronomy, NationalUniversity of Colombia, Bogotá, Colombia. E-mail: svmagnitskiy@unal.edu.co

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Mycorrhizas in Rooting of the Colombian Blueberry 373

INTRODUCTION

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

Vegetative propagation by stem cuttings is traditionally used to multiplyvarious Vaccinium sp. plants (Fischer et al., 2008; Litwinczuk et al., 2005;Morrison et al., 2000) and may be equally employed for the Colombianblueberry (Castrillón et al., 2008); the disadvantages of this method couldinclude slow rates of rhizogenesis and low rooting percentage. Other prop-agation techniques, such as by seeds, have different shortcomings, includ-ing slow germination rate, seed dormancy, and low uniformity of seedlingemergence (Magnitskiy and Ligarreto, 2007). Micropropagation of Vaccin-ium sp. has distinct advantages over propagation by cuttings in terms ofrooting percentage and plantlets survival (Meiners et al., 2007; Morrison et al.,2000), but it is not currently available to the growers of Andean region. Forvegetative propagation of Vaccinium sp., rooting hormones are commonlyapplied to increase rooting percentage in stem cuttings, whereas the effi-ciency of the hormone application is highly variable depending on plantspecies, 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 orrhizome cuttings for propagation of Vaccinium sp. At the same time, therooting of stolons and rhizomes constitutes a common way of propagationof the Colombian blueberry and other shrubs and creeping herbs undernatural conditions of a tropical forest. Rooting of rhizomes and stolonsrepresents a competitive strategy for rapid establishment of Vaccinium sp.plants under conditions of reforestation (Moola and Mallik, 1998) and, whilethe enhanced rhizome formation may not necessarily contribute to

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374 R. A. Ávila Díaz-Granados et al.

increased fruit production (Jamieson and Nickerson, 2003), it explains thepioneering role of the Colombian blueberry during revegetation of a tropi-cal mountain forest (McDonald et al., 2003). The use of stolon cuttings, iftechnically sustained, might serve as an additional method for vegetativepropagation of the Colombian blueberry in commercial nurseries.

A factor that apparently influences growth of the Colombian blueberryis the association of this plant with mycorrhizal fungi. Under natural condi-tions of the Colombian highlands, the Colombian blueberry is frequentlyfound as an understory shrub in oak forests of low light intensity, soils lowin nitrogen, and characterized by pH 5.0–5.5 and high presence of mycor-rhizas. Mycorrhizas of the Ericaceae are known to improve plant nutritionwith P, N, K, S, and Cu (Smith and Read, 2008; Yang et al., 2002) and toincrease stress tolerance in plants (Cairney and Meharg, 2003). However,there is a lack of published data on the importance of mycorrhizal associa-tions for the growth and propagation of the Colombian blueberry. In propa-gation practice, the rhizogenesis of unrooted cuttings may be affected bythe presence or absence of mycorrhizal fungi in the rooting media (Davies,2000). The capacity of the Colombian blueberry cuttings to form adventi-tious 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 thepresence of mycorrhizas in substrate might be expected.

Previous studies suggested the potential benefits of treating stem cuttingsof 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-mineralsubstrates (Castrillón et al., 2008). The purpose of the present study was toevaluate the effect of mycorrhizal fungi, when present in the substrate, onrhizogenesis of stem and stolon cuttings of the Colombian blueberry. Thecapacity of the Colombian blueberry cuttings to form adventitious roots inmycorrhiza-enriched substrate under the presence of rooting hormones wasalso 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 inthe municipality of Guachetá, Colombia (5.23° N, 73.41° W, 2,688 m oversea level, 14°C average annual temperature) and transported to the Facultyof Agronomy, National University of Colombia, Bogotá campus. The experi-ments 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 averagenight temperature), where propagation material (stem and stolon cuttings)

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Mycorrhizas in Rooting of the Colombian Blueberry 375

taken from homogenous plants with good vigor and without visible nutri-tional or pathological disorders was used.

Stem softwood cuttings (4-cm average length) having four to five greenleaves and the apical bud, and stolon tip cuttings (8-cm average length)consisting of five to six phytomers and the apical bud were used. Immedi-ately after collection, the basal ends of each cutting (approximately 1 cm)were moistened with distilled water and dipped into a rooting pastecontaining technical talc and 0, 200, or 400 mg L−1 chemically pure gradeIBA or NAA according to Castrillón et al. (2008). This protocol allowedusing lower concentrations of hormones for successful rooting of Vacciniumsp. rather than immersing cutting bases in highly concentrated solutions ofhormones (Fischer et al., 2008). Excess paste was tapped off after 20 mindrying, the cuttings were set into 3,000 cm3 plastic containers filled with 1:1v/v mixtures of peat:coal ash (mycorrhiza-free substrate) or mycorrhiza:coalash (mycorrhiza-enriched substrate) and arranged on a greenhouse benchunder an intermittent mist. The peat employed to prepare a mycorrhiza-freesubstrate was Base Substrate Klasmann® (Klasmann-Deilmann, Geeste,Germany). The peat-based commercial product used for inoculation withmycorrhizas was Micorrizafer® (Agrosafer Company, Medellín, Colombia)containing mycorrhiza propagules (spores, mycelium, and old colonizedroots) of a mixture of four mycorrhizal genera (Glomus, Entrophospora,Scutellospora, and Acaulospora) with minimal concentration of 230 sporesper gram of substrate. The pH was 5.5 in both substrates. The stem cuttingswere inserted vertically in the rooting media, while stolon cuttings wereplaced horizontally on the surface of the substrates. Relative humidity in thegreenhouse was maintained between 90% and 95%. Plastic black mesh wasadditionally established over the top of the greenhouse bench to provide50% shading.

Treatment plots were arranged in a completely randomized design overthe greenhouse bench. Each treatment plot consisted of a row of five stemcuttings or four stolon cuttings per ten treatments, and each treatment wasreplicated four times per trial, making 200 or 160 cuttings of stems or stolons,respectively. After a 60-day growth period, the cuttings were removed fromcontainers, and the roots were carefully washed to remove all substrateparticles. The number of viable cuttings, number of roots, and root lengthwere measured and rooting percentage per number of viable cuttings wascalculated 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 werecleared using hot 10% KOH in a water bath at 90°C during 20 min, washed

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376 R. A. Ávila Díaz-Granados et al.

thoroughly with distilled water three times, and immersed in 10% HCl for 15min 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 trypanblue:95 lactic acid v/v) at 90°C. The stain was prepared by mixing withdistilled water, glycerin, and lactic acid in proportions of 1:1:1 v/v/v. Thisstep was done without application of heat, but the incubation period wasextended to 12 h. The stain was poured off into another container, and theroots were placed in water-glycerin mix (50%). Following this treatment, thepercentage of the root length colonized by mycorrhizal fungi was detectedusing the gridline intersect technique (Giovannetti and Mosse, 1980).

Statistical Analysis

Analysis of variance (ANOVA) procedures were used to test for significanteffects 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 allstatistical tests. Significant differences between means for each group ofcuttings were estimated using the Duncan Multiple Range Test, p = 5%. Thecloseness of relationship among selected variables was determined bycorrelation analyses using the Pearson linear correlation coefficients (SASInstitute, Inc., 1996).

RESULTS AND DISCUSSION

Stem Cuttings

Stem cuttings treated with 200 or 400 mg L−1 IBA and placed in a substratewith no mycorrhiza added had an increment in viability compared torespective water-treated controls, although high viability rates were notnecessarily associated with high rooting percentage at day 60 (Table 1). Whengrowing in mycorrhiza-free substrate, cuttings had a higher rooting percent-age, up to 68.8% or 73.3% when treated with paste containing 200 mg L−1 IBAor 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 inmycorrhiza-free substrate significantly increased root number and rootlength as compared with controls (Table 1). The usage of 400 mg L−1 IBA orNAA did not contribute to higher average number of roots or root length(Table 1) in accordance with the previous data, suggesting that the applica-tion of IBA or NAA in rates that exceeded 200 mg L−1 do not increase root-ing in stem cuttings of the Colombian blueberry (Castrillón et al., 2008).

When plants were propagated in mycorrhiza-enriched substrate, viabil-ity of cuttings in control treatments (no hormone applied) was higher (95%)than that in mycorrhiza-free substrate (Table 1). At the same time, rootingpercentage was reduced in control cuttings placed in mycorrhiza-enriched

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TA

BLE

1Effec

t of M

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sence

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Rootin

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Va

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Rootin

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Via

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Rootin

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gs)

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of

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per

cuttin

gA

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cm)

−Myc

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−Myc

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H2O

(co

ntrol)

71 a

b95

a60

.2 a

b21

.0 b

1.8

c0.

2 b

3.15

bc

0.72

c

IBA

200

mg

L−180

a64

b68

.8 a

38.5

a6.

0 a

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a8.

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3.30

a40

0 m

g L−1

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ab

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2 b

0.8

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0.75

c

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8 ab

2.2

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lev

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ulti

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Ran

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378 R. A. Ávila Díaz-Granados et al.

substrate (21%) as compared with those growing in mycorrhiza-free sub-strate (60.2%).

These results indicated that the presence of mycorrhiza fungi in a sub-strate was beneficial for maintenance of cutting viability, whereas percent-age rooting, average number of roots, and root length were reduced. It iswell 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 theinfluence of mycorrhizal fungi on rhizogenesis of cuttings is not alwayspositive (Lanthier, 2006; Repac, 1998). It may be speculated that, in ourexperiment, the allocation of plant assimilates to the hyphal growth com-peted with the allocation of carbohydrates to the secondary root formation(Johnson et al., 1997; Smith and Read, 2008), and as a result, a negativeeffect of mycorrhizal fungi on the rhizogenesis was observed. The allocationof plant assimilates to the fungi during the establishment of a mycorrhizalassociation is well studied (Jakobsen and Rosendahl, 1990), while a similarprocess occurs during the establishment of a less mutual relationship for theplant (Johnson et al., 1997; Smith and Read, 2008). In the latter case, espe-cially under low levels of available nutrients in a substrate, mycorrhizalfungi might become parasitic (Johnson et al., 1997) and maintain viability ofcuttings, while exploring plant-derived metabolites and, thus, limitingformation of adventitious roots. It may be argued, however, that the inhibi-tion of root formation attributed to the presence of mycorrhizas in ourexperiment was short-termed and due to the lag-phase, a temporally inhibi-tion of root growth at the initial stages of colonization (Smith and Read,2008). Additionally, the absence of roots in cuttings would delay the forma-tion of mycorrhizal association. However, while the Colombian blueberry isa plant recalcitrant to vegetative propagation, approximately 40 days areusually sufficient for the cuttings to initiate development of adventitiousroots (Ligarreto and Magnitskiy, 2008 unpublished data). It is also worthnoticing that a level of genotype-genotype specificity between the host andthe fungus defines a range of responses in a mutualism-parasitism contin-uum, ranging from a response negative for the plant to a positive one, espe-cially in the case of Vaccinium sp. plants, for which few commercialproducts containing specialized mycorrhizal fungi are available (Lanthier,2006; Vega et al., 2009).

With hormones applied to the cutting base in mycorrhiza-enriched sub-strate, both cutting viability and rooting did not exceed 65% and 50%,respectively (Table 1). The presence of mycorrhiza had a more adverseeffect on rooting for water-treated cuttings (21%) than for hormone-treatedones. When IBA or NAA were applied to the cutting base, the negativeeffect of mycorrhizas on rooting percentage was less noticeable, resulting inup to 41.7% or 50% rooting, respectively. These antagonistic effects ofhormones and mycorrhizas on rhizogenesis in mycorrhiza-enriched

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Mycorrhizas in Rooting of the Colombian Blueberry 379

substrate may be explained from the point of view that hormones, such asIBA or NAA, when present in substrate, had a negative affect on a fungusgrowth (Lanthier, 2006) or signaling between the fungi and the plant.

In summary, stem cuttings grown in mycorrhiza-enriched substrate hadlower rooting percentage, reduced number of roots, and shorter rootscompared to their counterparts grown in mycorrhiza-free substrate. Treatingcuttings with 200 mg L−1 IBA or NAA in mycorrhiza-enriched substratesignificantly increased root number and root length compared to that ofcontrol cuttings. A similar trend was noticed for cuttings in mycorrhiza-freesubstrate. However, for most treatments, both root number and root lengthwere 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 orexceeded 50% in both substrates (Table 2). Mycorrhizal colonization was, ingeneral, lower in controls (water-treated) than in hormone-treated cuttingsfor both mycorrhiza-enriched and mycorrhiza-free substrates (Table 2). Thehigh rates of root length colonization in substrate with no external mycor-rhizas added (Table 2) may be explained by the initial presence of fungi ina nonsterilized substrate.

In the substrate with mycorrhiza added, strong correlations werefound among cutting viability or rooting percentage, on the one side, andpercentage of colonization, on the other side. For hormone-treated cut-tings (0, 200, and 400 mg L−1) in mycorrhiza-enriched substrate, the nega-tive correlations among cutting viability and percentage colonization werefound: −0.89 for IBA and −0.54 for NAA. The positive correlations amongrooting percentage and percentage of colonization for the same treatmentswere 0.91 for IBA and 0.80 for NAA. In other words, increasing rates ofhormones were associated with both high rooting percentage and highcolonization of roots, if the latest were formed. However, in general,

TABLE 2 Colonization of Roots of Stem Cuttings of the Colombian Blueberry(Vaccinium meridionale Swartz) Treated with Rooting Hormones withMycorrhizal 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 ab400 mg L−1 50.0 b 80.3 a

NAA200 mg L−1 57.1 ab 75.0 ab400 mg L−1 60.0 ab 90.0 a

Treatment means in the same column followed by the same letter are not significantlydifferent at 5% level of Duncan Multiple Range Test.

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380 R. A. Ávila Díaz-Granados et al.

reduced root formation was observed in mycorrhiza-enriched substrate(Table 1) and this effect of fungi was less noticeable when increasing ratesof IBA or NAA (Table 1).

Stolon Cuttings

The data on viability and rooting for stolon cuttings were much less consis-tent than that for stem cuttings (Table 3), which makes interpretation of theresults difficult. In various treatments, the stolon cuttings were dried off byday 30 from the start of the experiment. The pattern in viability in mycor-rhiza-enriched substrate at day 60 after planting was similar for both stolonand stem cuttings; more viable cuttings were registered when no hormoneswere 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 viableup to the end of the experiment but did not produce roots. When treatedwith IBA or NAA, the viability of stolon cuttings was, similarly to thatobserved in stem cuttings (Table 1), higher in mycorrhiza-free substrate andlower 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 sub-strate produced roots.

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

Stolon cuttings may be successfully used for vegetative propagation ofvarious species, including white clover (Annicchiarico and Piano, 2004;Murray et al., 2002) and strawberry (Smith, 1972), and application of rootinghormones 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 cuttingsof goldenrod (Solidago sp.) increased rooting percentage and contributed toproduction of vigorous adventitious roots. Treating the base of stoloncuttings of a medicinal herb Picrorhiza kurrooa (Royle ex Benth) with100–200 mg L−1 IAA or IBA increased percentage survival of cuttings andreduced the number of days required for rooting; the effect was proved ontop segments of stolon cuttings but not on the basal segments (Nautiyalet al., 2001).

The plants of the genus Vaccinium present a long-term persistence inthe understory of many second-growth forests under extremely low lightconditions that favor storage of photosynthates in plagiotropic shoots androots at the expense of above-ground stems and foliage (Alaback and

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TA

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Myc

orr

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ubst

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Stolo

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Ave

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−Myc

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382 R. A. Ávila Díaz-Granados et al.

Tappeiner, 1991; Moola and Mallik, 1998), suggesting the possible use ofstolons or rhizomes for propagation practices. Low viability and poor root-ing performance of stolon cuttings in our study might be due to the exces-sive light conditions, inadequate age (softwood) or length of cuttings,insufficient number of vegetative buds or bud dormancy, or inadequatehormone concentration in hormone-treated cuttings, among others. It mayalso be that fungal (Cline, 2004) or bacterial (Kobayashi et al., 1995) infec-tions typical for Vaccinium sp., while not visually observed in our study,decreased viability of fresh stolon cuttings when these were placed in acontact with substrates.

According to our results, the use of vegetative stem cuttings is a relativelyfast and simple method for propagation of the Colombian blueberry, despitethe fact that Vaccinium sp. cuttings have low rates of adventitious root devel-opment (Litwinczuk et al., 2005; Miller et al., 2006). The presence of mycor-rhizas and the use of a nonsterile substrate, in general, contributed to reducedroot development on stem cuttings of the Colombian blueberry. While usinghormones may diminish the effects of fungi on root development in cuttingsof the Colombian blueberry, a sterilization of rooting media might be furthersuggested to increase the rates of rhizogenesis in unrooted cuttings. In futureexperiments, the benefits of inoculation of previously rooted stem cuttingswith mycorrhizal fungi, as well as changing the nutrient levels in the rootingmedia or inoculation rates, may be addressed. The use of stolon cuttings forvegetative propagation of the Colombian blueberry has low practical applica-bility. The results of the study might further serve to establish the optimal sub-strate conditions and source of stem cuttings for vegetative propagation of theColombian blueberry in commercial culture.

ACKNOWLEDGMENTS

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

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