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Mycorrhizal inoculation enhances growth and nutrient uptake of micropropagated apple rootstocks during weaning in commercial substrates of high nutrient availability
Applied Soil Ecology 15 (2000) 113–118
Mycorrhizal inoculation enhances growth and nutrient uptake of micropropagated apple rootstocks during weaning in commercial substrates of high nutrient availability A. Schubert∗ , G. Lubraco Dipartimento Colture arboree, Universita’ di Torino, via Leonardo da Vinci 44, 10095 Grugliasco (TO), Italy Received 31 May 1999; received in revised form 1 December 1999; accepted 23 March 2000
Abstract Apple (Malus pumila L.) plants of the rootstock clone MM106 were micropropagated from axillary buds and, after rooting, were transplanted into pots containing three different commercial-type peat-based substrates, fertilized before planting with about 100 mg soluble P per liter (about 300 mg kg−1 wet substrate). Plants were inoculated at transplant with the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe. After 112 days of growth, roots were heavily colonized by mycorrhizal fungi. Inoculation increased P uptake in all substrates. Plant growth was enhanced by inoculation in two of the substrates. During the growth period, the P content of the substrate was severely depleted, and this may explain the intense root colonization and nutrient uptake enhancement observed in such nutrient-rich substrates. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Phosphorus; Malus; Glomus; Plant propagation
1. Introduction In vitro culture of fruit plants is an established method of plant propagation, now applied very commonly because of advantages such as the absence of pathogens and the high numbers of plants which can be obtained in a relatively short time and reduced space. Clonal apple rootstocks are routinely used in apple orchard plantings in the world and are mostly obtained by micropropagation (Zimmermann, 1991). Inoculation with arbuscular mycorrhizal (AM) fungi of micropropagated fruit plants at transplant ex vitro improves growth and nutrient uptake during ∗ Corresponding author. Tel.: +39-011-6708654; fax: +39-011-6708658. E-mail address: [email protected] (A. Schubert)
the weaning stage, yielding plants of larger size and improved commercial characteristics (Lovato et al., 1992; Cordier et al., 1996). The apple plant is commonly infected by AM fungi (Koch et al., 1981), and apple plants grown in soils of low or high nutrient availability show significant growth enhancements if inoculated with AM fungi (Plenchette et al., 1981; Morin et al., 1994). Although the effects of AM inoculation on micropropagated apple plants in mineral soils are relatively well known, little information is available on the effects of AM inoculation in the artificial substrates which are used in commercial weaning of micropropagated plants. Commercial fruit plant micropropagation normally makes use of peat-based substrates, containing no mineral soil, enriched in N, P, and K (Preece and Sutter, 1991). The high chemical fertility of these
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substrates is designed to improve plant growth, but it can have negative effects on the growth of the mycorrhizal fungi, which show slow development and little infection potential in soils of high P content (Smith and Read, 1997). In this work, we studied the development of the fungus and of the plant partner in apple plants inoculated with AM fungi. Inoculation and weaning were carried out in pots containing commercial growth substrates.
2. Materials and methods
Table 1 Chemical analysis of the substrates used in the experiment (PT: commercial peat-based, fertilized substrate; PE: PT substrate/perlite 60/40 v/v and TC: acid sphagnum peat amended with a slow-release fertilizer) Substrate
PT
PE
TC
pH Organic matter (%) Organic C (%) Total N (mg kg−1 (wet))a Total P (mg kg−1 (wet)) Soluble P added at substrate preparation (mg kg−1 (wet))b Bulk densityc (dry)d (g l−1 ) Bulk density (wet) (g l−1 )
6.4 82.1 47.6 2436.8 419.6 306.0
6.4 58.4 37.9 1730.3 399.5 294.8
6.7 81.6 45.5 1889.9 442.6 298.9
192.7 349.6
133.5 217.7
189.7 394.4
a
Apple (Malus pumila L.) rootstocks (clone MM106) were micropropagated from axillary buds. Buds were sampled from surface-sterilized young shoots and were cultured for 60 days in a proliferation medium containing Murashige and Skoog (MS) salts and vitamins, and the following hormones: indolebutyric acid 0.1 mg l−1 , benzylaminopurine 1 mg l−1 and GA3 0.1 mg l−1 . After proliferation, single shoots were subcultured for 30 days into a rooting medium containing MS salts and vitamins, and indoleacetic acid 2 mg l−1 . All media contained 30 g l−1 sucrose and 6% agar. After rooting (about four roots per plant, about 15 mm long) plants were transplanted into 7 cm×7 cm, 280 ml plastic pots. Transplant was made in conditions of high ambient humidity to reduce damage to the plantlets. At transplant, pots were filled with different substrates, composed of mixtures of a peat-based commercial substrate (TRIOHUM Potgrond P, Klasmann–Deilmann GmbH, Geeste, Germany), acid sphagnum peat (SF 40101, Juaskvea, Finland) and perlite (Agripan 100, Italy). The substrates were named PT (Potgrond P 100%), PE (Potgrond P/perlite 60/40 v/v) and TC (acid sphagnum peat, added at pot filling with 3 g l−1 solid Ca(OH)2 to reach pH 6.7, and with 3 g l−1 of a fertilizer (Nitrophoska Gold), containing 15% slow-release N, 9% P2 O5 , 15% K, and 2% Mg). Substrates were wetted before filling the pots. The main characteristics of the substrates are presented in Table 1. At transplant, plants were inoculated with the AM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe (isolate BEG 12), in the form of a mixture of spores, soil, and infected clover roots. Ten grams of inoculum were placed in each planting hole about
Wet: substrate added with water to soil capacity. In substrates PT and PE soluble P was added at substrate preparation by the producer, in TC it was added at pot filling. c Density measurements were made on substrate subjected to the same compression as in pots. d Dry: substrate dried at 105◦ C. b
1 cm below the roots, for a total of 80 propagules per plant, measured with the most probable number method (Porter, 1979). Non-inoculated roots received 10 g of the autoclaved soil used for inoculum production (Olsen P 8 mg kg−1 ). Plants were grown in a partially climate-controlled greenhouse, with day temperature ranging from 15 to 30◦ C. Pots were given daily 10 mm irrigation water, in order to keep the substrate water content close to field capacity. After transplant, plants were covered with a transparent plastic sheet to maintain air relative humidity at 100%. One week after transplant, the cover was progressively removed to allow plants to acclimate to ambient conditions. Plant growth (height and total leaf area) was monitored weekly from 5 June to 25 September (112 days). At the end of the experiment, plants were collected and weighed. Roots were stained to assess mycorrhizal colonization using the method of Giovannetti and Mosse (1980). The shoots and the substrates were dried at 105◦ C. The total P concentration of plants and substrates was assessed using standard analytical techniques (Page et al., 1982). The amount of P in the plants at the end of the experiment was estimated by applying to the roots the same percent of P content and the same dry weight/fresh weight ratio as measured in shoots. Each treatment consisted of 48 replicate plants; the P
A. Schubert, G. Lubraco / Applied Soil Ecology 15 (2000) 113–118
content per treatment was assessed on two replicate pools of dried shoots and substrates from 24 pots. The experiment was set up with a randomized block design, each block containing six replicate plants per treatment, for a total of eight blocks. Data were processed by analysis of variance, and averages were separated by the Duncan test.
3. Results 3.1. Root mycorrhizal colonization and plant growth At the end of the experiment the roots of noninoculated plants were not colonized by the AM fungus. Inoculated plants were mycorrhizal, and root colonization was (averages±S.E.): 72.8±1.5% in the substrate PT, 84.0±2.2% in PE, and 82.5±3.2% in TC.
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Plant survival after transplant was 96% on an average, with no significant differences among treatments. At the end of the experiment, shoot length and total leaf area were significantly higher in the substrates PT and TC, which had higher nutrient content per pot, due to the low bulk density of the PE substrate. Inoculation induced an increase in shoot length and total leaf area in the PE and TC substrates (significantly in the latter case), as compared to the non-inoculated plants. The opposite was true in the PT substrate, where non-inoculated plants were significantly larger than mycorrhizal plants (Fig. 1). Similar results were given by the measurement of the shoot and root fresh weight; in this case inoculation significantly increased plant growth only in the case of the TC substrate (Fig. 2). The plant root/shoot fresh weight ratio was overall higher in the TC substrate. The ratio was not significantly affected by inoculation in the PE and TC
Fig. 1. Shoot length (above) and total leaf area (below) of micropropagated apple plants, inoculated with the AM fungus Glomus mosseae (MYC) or non-inoculated (C) in three different substrates (PT, PE and TC) during the weaning stage. Values marked by the same letters do not differ significantly at p=0.05.
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Fig. 2. Shoot and root fresh weight (above) and root/shoot fresh weight ratio (below) of micropropagated apple plants, inoculated with the AM fungus Glomus mosseae (MYC) or non-inoculated (C) in three different substrates (PT, PE and TC) during the weaning stage. Values marked by the same letters do not differ significantly at p=0.05.
substrates, while it was significantly lower in mycorrhizal plants in the PT substrate (Fig. 2). 3.2. Plant and soil nutrient content Fungal inoculation significantly affected the P content of plants, which was higher in mycorrhizal than in non-inoculated plants in all substrates, the difference being significant in substrates PT and PE (Fig. 3). Plants grown on the TC substrate had significantly lower P contents than plants grown on the other substrates. The total P content of the substrates decreased during the experiment: 112 days after transplant, in inoculated pots total P content was reduced, on an average of the three substrates, by about 94%, and only about 7% of the lost P was found in the plant tissues. In non-inoculated pots about 32% P was recovered in the substrate after plant cultivation, and 6% was found in the plant tissues (Table 2).
4. Discussion The results of this experiment show that inoculation with AM fungi induced root fungal colonization and increased nutrient content of apple micropropagated plants in three commercial, nutrient-rich substrates. Most of the published experiments on inoculation of micropropagated plants have been carried out in substrates relatively poor in nutrients, with available P contents not higher than 100 mg kg−1 . In the case of apple, Morin et al. (1994) inoculated micropropagated plants in a mineral soil rich in P, which however had physical and chemical characteristics very different from those of a commercial, peat-based substrate. In the substrates we used, P was added, either by the producer or by us, as a soluble fertilizer at concentrations of about 100 mg l−1 (PT and TC), and 60 mg l−1 (PE), corresponding to about 300 mg kg−1 wet substrate. In spite of this high soluble P content, inoculated plants were heavily infected by AM fungi
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Fig. 3. Concentration of P in the dry shoots of micropropagated apple plants, inoculated with the AM fungus Glomus mosseae (MYC) or non-inoculated (C) in three different substrates (PT, PE and TC) during the weaning stage. Values marked by the same letters do not differ significantly at p=0.05.
and shoot P concentration was significantly higher in mycorrhizal than in non-mycorrhizal plants. Thus stable mycorrhizal inoculation can be obtained in such substrates, giving micropropagated plantlets the added values of increased nutrient content, increased growth, and potential defence against root pathogens. An intense root colonization and an enhancement of plant P content are usually not expected in soils of high soluble P content, as root infection by AM fungi is slowed down by soil P availability (Smith and Read, 1997). We measured the total P content of the substrate and of the plant at the beginning and at the end of the experiment. On an average only about 7% of the total P present in the pots was incorporated into the plants, and this percentage increases to about 10% of the soluble P present in the substrates as a fertilizer. However at the end of the experiment total P content per pot was much lower than at transplant, its values ranging
between 13 and 54 mg kg−1 wet substrate. Thus AM fungi and plant roots, originally introduced into a soil containing high amounts of soluble P, were exposed for part of the experiment to relatively low P concentrations. This can explain the high rate of root colonization and the increase in P content in mycorrhizal plants. From the measured concentrations of total P at the beginning and at the end of the experiment, and from the P measured in the plants, we can estimate P losses from the substrate. These losses are high (50–88% of the total P present in the substrate at the beginning of the experiment) and are probably due to the leaching by irrigation water. High irrigation is a common practice in order to avoid water stress during weaning of micropropagated plants, and also in our experiment 10 mm irrigation water were applied daily. It is interesting to note that the losses were much higher in
Table 2 Total P content per pot in three different substrates (PT, PE and TC), inoculated (MYC) or non-inoculated (C) with the AM fungus Glomus mosseae, at transplant and at the end of the experiment, total P content per plant at the end of the experiment, and estimated loss of P during plant cultivation (means±S.E.) Substrate
AM treatment
Total P per pot at transplant (mg)
Total P per pot at the end of the experiment (mg)
Total P per plant at the end of the experiment (mg)
Estimated loss of P per pot (mg)
PT PT PE PE TC TC
MYC C MYC C MYC C
41.1±0.12 41.1±0.12 24.3±0.26 24.3±0.26 48.9±0.33 48.9±0.33
2.9±0.15 15.2±0.23 0.6±0.05 3.7±0.05 4.0±0.07 21.5±0.28
2.5±0.32 5.3±0.27 2.1±0.26 0.8±0.06 3.3±0.40 1.4±0.17
35.7 20.6 21.6 19.8 41.6 25.9
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A. Schubert, G. Lubraco / Applied Soil Ecology 15 (2000) 113–118
inoculated pots, where more than the soluble P added at substrate preparation was lost from the soil than in control pots which lost nearly the same amount added as soluble fertilizer. The inoculation with mycorrhizal fungi has been shown to enhance, although indirectly, the solubilization of organic P (Joner and Jakobsen, 1994), and this may have provided more soluble P in inoculated pots, which could be leached by irrigation water. Mycorrhizal colonization and the enhancement of nutrient uptake increased growth of both roots and shoots only in two out of three substrates: in the substrate PT control plants grew more than inoculated plants. The growth enhancement of AM inoculation can be due to factors other than improved P uptake, such as improved water relations (Graham and Syvertsen, 1984) and protection against root pathogens (Linderman, 1994). Although inoculation seems in general to be effective to obtain micropropagated, mycorrhizal plants, it is not possible to easily predict whether a growth response can be obtained during weaning in a given commercial substrate. Acknowledgements The authors wish to thank Prof. E. Barberis for useful discussion on the interpretation of substrate analysis data. References Cordier, C., Trouvelot, A., Gianinazzi, S., Gianinazzi-Pearson, V., 1996. Arbuscular mycorrhiza technology applied to micropropagated Prunus avium and to protection against Phytophthora cinnamomi. Agronomie 16, 679–688.
Giovannetti, M., Mosse, B., 1980. An evaluation of techniques for measuring vesicular–arbuscular mycorrhizal infection of roots. New Phytol. 84, 489–500. Graham, J.H., Syvertsen, J.P., 1984. Influence of vesicular– arbuscular mycorrhiza on the hydraulic conductivity of roots of two Citrus rootstocks. New Phytol. 97, 277–284. Joner, E.J., Jakobsen, I., 1994. Contribution by two arbuscular mycorrhizal fungi to P uptake by cucumber (Cucumis sativus L.) from 32 P-labelled organic matter during mineralisation in soil. Plant Soil 163, 203–209. Koch, B.L., Covey, R.P., Larsen, H.J., 1981. Response of apple seedlings in fumigated soil to phosphorous and vesicular–arbuscular mycorrhiza. HortScience 17, 232–233. Linderman, R.G., 1994. Role of VAM in biocontrol. In: Pfleger, F.L., Linderman, R.G. (Eds.), Mycorrhizae and Plant Health. APS Press, St Paul, MN, pp. 1–26. Lovato, P., Guillemin, J.P., Gianinazzi, S., 1992. Application of commercial endomycorrhizal fungal inoculants to the establishment of micropropagated grapevine rootstock and pineapple plants. Agronomie 12, 873–880. Morin, F., Fortin, J.A., Hamel, C., Granger, R.L., Smith, D.L., 1994. Apple rootstock response to vesicular–arbuscular mycorrhizal fungi in a high phosphorous soil. J. Am. Soc. Hort. Sci. 119, 578–583. Page, A.L., Miller, R.H., Keeney, D.R., 1982. Methods of Soil Analysis. American Society of Agronomy, Madison, WI, 1158 pp. Plenchette, C., Furlan, V., Fortin, J.A., 1981. Growth stimulation of apple trees in unsterilized soils under field conditions with VA mycorrhiza inoculum. Can. J. Bot. 59, 2003–2008. Porter, W.M., 1979. The most probable number method for enumerating infective propagules of vesicular–arbuscular mycorrhizal fungi in soil. Aust. J. Soil Res. 17, 515–519. Preece, J.E., Sutter, E.G., 1991. Acclimatization of micropropagated plants to the greenhouse and the field. In: Debergh, P.C., Zimmermann, R.H. (Eds.), Micropropagation. Kluwer Academic Publishers, Dordrecht, pp. 71–93. Smith, S.E., Read, D.J., 1997. Mycorrhizal Symbiosis. Academic Press, San Diego, CA, 605 pp. Zimmermann, R.H., 1991. Micropropagation of temperate zone fruit and nut crops. In: Debergh, P.C., Zimmermann, R.H. (Eds.), Micropropagation. Kluwer Academic Publishers, Dordrecht, pp. 231–246.
Mycorrhizal inoculation enhances growth and nutrient uptake of micropropagated apple rootstocks during weaning in commercial substrates of high nutrient availability A. Schubert∗ , G. Lubraco Dipartimento Colture arboree, Universita’ di Torino, via Leonardo da Vinci 44, 10095 Grugliasco (TO), Italy Received 31 May 1999; received in revised form 1 December 1999; accepted 23 March 2000
Abstract Apple (Malus pumila L.) plants of the rootstock clone MM106 were micropropagated from axillary buds and, after rooting, were transplanted into pots containing three different commercial-type peat-based substrates, fertilized before planting with about 100 mg soluble P per liter (about 300 mg kg−1 wet substrate). Plants were inoculated at transplant with the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe. After 112 days of growth, roots were heavily colonized by mycorrhizal fungi. Inoculation increased P uptake in all substrates. Plant growth was enhanced by inoculation in two of the substrates. During the growth period, the P content of the substrate was severely depleted, and this may explain the intense root colonization and nutrient uptake enhancement observed in such nutrient-rich substrates. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Phosphorus; Malus; Glomus; Plant propagation
1. Introduction In vitro culture of fruit plants is an established method of plant propagation, now applied very commonly because of advantages such as the absence of pathogens and the high numbers of plants which can be obtained in a relatively short time and reduced space. Clonal apple rootstocks are routinely used in apple orchard plantings in the world and are mostly obtained by micropropagation (Zimmermann, 1991). Inoculation with arbuscular mycorrhizal (AM) fungi of micropropagated fruit plants at transplant ex vitro improves growth and nutrient uptake during ∗ Corresponding author. Tel.: +39-011-6708654; fax: +39-011-6708658. E-mail address: [email protected] (A. Schubert)
the weaning stage, yielding plants of larger size and improved commercial characteristics (Lovato et al., 1992; Cordier et al., 1996). The apple plant is commonly infected by AM fungi (Koch et al., 1981), and apple plants grown in soils of low or high nutrient availability show significant growth enhancements if inoculated with AM fungi (Plenchette et al., 1981; Morin et al., 1994). Although the effects of AM inoculation on micropropagated apple plants in mineral soils are relatively well known, little information is available on the effects of AM inoculation in the artificial substrates which are used in commercial weaning of micropropagated plants. Commercial fruit plant micropropagation normally makes use of peat-based substrates, containing no mineral soil, enriched in N, P, and K (Preece and Sutter, 1991). The high chemical fertility of these
0929-1393/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 9 - 1 3 9 3 ( 0 0 ) 0 0 0 8 6 - X
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substrates is designed to improve plant growth, but it can have negative effects on the growth of the mycorrhizal fungi, which show slow development and little infection potential in soils of high P content (Smith and Read, 1997). In this work, we studied the development of the fungus and of the plant partner in apple plants inoculated with AM fungi. Inoculation and weaning were carried out in pots containing commercial growth substrates.
2. Materials and methods
Table 1 Chemical analysis of the substrates used in the experiment (PT: commercial peat-based, fertilized substrate; PE: PT substrate/perlite 60/40 v/v and TC: acid sphagnum peat amended with a slow-release fertilizer) Substrate
PT
PE
TC
pH Organic matter (%) Organic C (%) Total N (mg kg−1 (wet))a Total P (mg kg−1 (wet)) Soluble P added at substrate preparation (mg kg−1 (wet))b Bulk densityc (dry)d (g l−1 ) Bulk density (wet) (g l−1 )
6.4 82.1 47.6 2436.8 419.6 306.0
6.4 58.4 37.9 1730.3 399.5 294.8
6.7 81.6 45.5 1889.9 442.6 298.9
192.7 349.6
133.5 217.7
189.7 394.4
a
Apple (Malus pumila L.) rootstocks (clone MM106) were micropropagated from axillary buds. Buds were sampled from surface-sterilized young shoots and were cultured for 60 days in a proliferation medium containing Murashige and Skoog (MS) salts and vitamins, and the following hormones: indolebutyric acid 0.1 mg l−1 , benzylaminopurine 1 mg l−1 and GA3 0.1 mg l−1 . After proliferation, single shoots were subcultured for 30 days into a rooting medium containing MS salts and vitamins, and indoleacetic acid 2 mg l−1 . All media contained 30 g l−1 sucrose and 6% agar. After rooting (about four roots per plant, about 15 mm long) plants were transplanted into 7 cm×7 cm, 280 ml plastic pots. Transplant was made in conditions of high ambient humidity to reduce damage to the plantlets. At transplant, pots were filled with different substrates, composed of mixtures of a peat-based commercial substrate (TRIOHUM Potgrond P, Klasmann–Deilmann GmbH, Geeste, Germany), acid sphagnum peat (SF 40101, Juaskvea, Finland) and perlite (Agripan 100, Italy). The substrates were named PT (Potgrond P 100%), PE (Potgrond P/perlite 60/40 v/v) and TC (acid sphagnum peat, added at pot filling with 3 g l−1 solid Ca(OH)2 to reach pH 6.7, and with 3 g l−1 of a fertilizer (Nitrophoska Gold), containing 15% slow-release N, 9% P2 O5 , 15% K, and 2% Mg). Substrates were wetted before filling the pots. The main characteristics of the substrates are presented in Table 1. At transplant, plants were inoculated with the AM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe (isolate BEG 12), in the form of a mixture of spores, soil, and infected clover roots. Ten grams of inoculum were placed in each planting hole about
Wet: substrate added with water to soil capacity. In substrates PT and PE soluble P was added at substrate preparation by the producer, in TC it was added at pot filling. c Density measurements were made on substrate subjected to the same compression as in pots. d Dry: substrate dried at 105◦ C. b
1 cm below the roots, for a total of 80 propagules per plant, measured with the most probable number method (Porter, 1979). Non-inoculated roots received 10 g of the autoclaved soil used for inoculum production (Olsen P 8 mg kg−1 ). Plants were grown in a partially climate-controlled greenhouse, with day temperature ranging from 15 to 30◦ C. Pots were given daily 10 mm irrigation water, in order to keep the substrate water content close to field capacity. After transplant, plants were covered with a transparent plastic sheet to maintain air relative humidity at 100%. One week after transplant, the cover was progressively removed to allow plants to acclimate to ambient conditions. Plant growth (height and total leaf area) was monitored weekly from 5 June to 25 September (112 days). At the end of the experiment, plants were collected and weighed. Roots were stained to assess mycorrhizal colonization using the method of Giovannetti and Mosse (1980). The shoots and the substrates were dried at 105◦ C. The total P concentration of plants and substrates was assessed using standard analytical techniques (Page et al., 1982). The amount of P in the plants at the end of the experiment was estimated by applying to the roots the same percent of P content and the same dry weight/fresh weight ratio as measured in shoots. Each treatment consisted of 48 replicate plants; the P
A. Schubert, G. Lubraco / Applied Soil Ecology 15 (2000) 113–118
content per treatment was assessed on two replicate pools of dried shoots and substrates from 24 pots. The experiment was set up with a randomized block design, each block containing six replicate plants per treatment, for a total of eight blocks. Data were processed by analysis of variance, and averages were separated by the Duncan test.
3. Results 3.1. Root mycorrhizal colonization and plant growth At the end of the experiment the roots of noninoculated plants were not colonized by the AM fungus. Inoculated plants were mycorrhizal, and root colonization was (averages±S.E.): 72.8±1.5% in the substrate PT, 84.0±2.2% in PE, and 82.5±3.2% in TC.
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Plant survival after transplant was 96% on an average, with no significant differences among treatments. At the end of the experiment, shoot length and total leaf area were significantly higher in the substrates PT and TC, which had higher nutrient content per pot, due to the low bulk density of the PE substrate. Inoculation induced an increase in shoot length and total leaf area in the PE and TC substrates (significantly in the latter case), as compared to the non-inoculated plants. The opposite was true in the PT substrate, where non-inoculated plants were significantly larger than mycorrhizal plants (Fig. 1). Similar results were given by the measurement of the shoot and root fresh weight; in this case inoculation significantly increased plant growth only in the case of the TC substrate (Fig. 2). The plant root/shoot fresh weight ratio was overall higher in the TC substrate. The ratio was not significantly affected by inoculation in the PE and TC
Fig. 1. Shoot length (above) and total leaf area (below) of micropropagated apple plants, inoculated with the AM fungus Glomus mosseae (MYC) or non-inoculated (C) in three different substrates (PT, PE and TC) during the weaning stage. Values marked by the same letters do not differ significantly at p=0.05.
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Fig. 2. Shoot and root fresh weight (above) and root/shoot fresh weight ratio (below) of micropropagated apple plants, inoculated with the AM fungus Glomus mosseae (MYC) or non-inoculated (C) in three different substrates (PT, PE and TC) during the weaning stage. Values marked by the same letters do not differ significantly at p=0.05.
substrates, while it was significantly lower in mycorrhizal plants in the PT substrate (Fig. 2). 3.2. Plant and soil nutrient content Fungal inoculation significantly affected the P content of plants, which was higher in mycorrhizal than in non-inoculated plants in all substrates, the difference being significant in substrates PT and PE (Fig. 3). Plants grown on the TC substrate had significantly lower P contents than plants grown on the other substrates. The total P content of the substrates decreased during the experiment: 112 days after transplant, in inoculated pots total P content was reduced, on an average of the three substrates, by about 94%, and only about 7% of the lost P was found in the plant tissues. In non-inoculated pots about 32% P was recovered in the substrate after plant cultivation, and 6% was found in the plant tissues (Table 2).
4. Discussion The results of this experiment show that inoculation with AM fungi induced root fungal colonization and increased nutrient content of apple micropropagated plants in three commercial, nutrient-rich substrates. Most of the published experiments on inoculation of micropropagated plants have been carried out in substrates relatively poor in nutrients, with available P contents not higher than 100 mg kg−1 . In the case of apple, Morin et al. (1994) inoculated micropropagated plants in a mineral soil rich in P, which however had physical and chemical characteristics very different from those of a commercial, peat-based substrate. In the substrates we used, P was added, either by the producer or by us, as a soluble fertilizer at concentrations of about 100 mg l−1 (PT and TC), and 60 mg l−1 (PE), corresponding to about 300 mg kg−1 wet substrate. In spite of this high soluble P content, inoculated plants were heavily infected by AM fungi
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Fig. 3. Concentration of P in the dry shoots of micropropagated apple plants, inoculated with the AM fungus Glomus mosseae (MYC) or non-inoculated (C) in three different substrates (PT, PE and TC) during the weaning stage. Values marked by the same letters do not differ significantly at p=0.05.
and shoot P concentration was significantly higher in mycorrhizal than in non-mycorrhizal plants. Thus stable mycorrhizal inoculation can be obtained in such substrates, giving micropropagated plantlets the added values of increased nutrient content, increased growth, and potential defence against root pathogens. An intense root colonization and an enhancement of plant P content are usually not expected in soils of high soluble P content, as root infection by AM fungi is slowed down by soil P availability (Smith and Read, 1997). We measured the total P content of the substrate and of the plant at the beginning and at the end of the experiment. On an average only about 7% of the total P present in the pots was incorporated into the plants, and this percentage increases to about 10% of the soluble P present in the substrates as a fertilizer. However at the end of the experiment total P content per pot was much lower than at transplant, its values ranging
between 13 and 54 mg kg−1 wet substrate. Thus AM fungi and plant roots, originally introduced into a soil containing high amounts of soluble P, were exposed for part of the experiment to relatively low P concentrations. This can explain the high rate of root colonization and the increase in P content in mycorrhizal plants. From the measured concentrations of total P at the beginning and at the end of the experiment, and from the P measured in the plants, we can estimate P losses from the substrate. These losses are high (50–88% of the total P present in the substrate at the beginning of the experiment) and are probably due to the leaching by irrigation water. High irrigation is a common practice in order to avoid water stress during weaning of micropropagated plants, and also in our experiment 10 mm irrigation water were applied daily. It is interesting to note that the losses were much higher in
Table 2 Total P content per pot in three different substrates (PT, PE and TC), inoculated (MYC) or non-inoculated (C) with the AM fungus Glomus mosseae, at transplant and at the end of the experiment, total P content per plant at the end of the experiment, and estimated loss of P during plant cultivation (means±S.E.) Substrate
AM treatment
Total P per pot at transplant (mg)
Total P per pot at the end of the experiment (mg)
Total P per plant at the end of the experiment (mg)
Estimated loss of P per pot (mg)
PT PT PE PE TC TC
MYC C MYC C MYC C
41.1±0.12 41.1±0.12 24.3±0.26 24.3±0.26 48.9±0.33 48.9±0.33
2.9±0.15 15.2±0.23 0.6±0.05 3.7±0.05 4.0±0.07 21.5±0.28
2.5±0.32 5.3±0.27 2.1±0.26 0.8±0.06 3.3±0.40 1.4±0.17
35.7 20.6 21.6 19.8 41.6 25.9
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inoculated pots, where more than the soluble P added at substrate preparation was lost from the soil than in control pots which lost nearly the same amount added as soluble fertilizer. The inoculation with mycorrhizal fungi has been shown to enhance, although indirectly, the solubilization of organic P (Joner and Jakobsen, 1994), and this may have provided more soluble P in inoculated pots, which could be leached by irrigation water. Mycorrhizal colonization and the enhancement of nutrient uptake increased growth of both roots and shoots only in two out of three substrates: in the substrate PT control plants grew more than inoculated plants. The growth enhancement of AM inoculation can be due to factors other than improved P uptake, such as improved water relations (Graham and Syvertsen, 1984) and protection against root pathogens (Linderman, 1994). Although inoculation seems in general to be effective to obtain micropropagated, mycorrhizal plants, it is not possible to easily predict whether a growth response can be obtained during weaning in a given commercial substrate. Acknowledgements The authors wish to thank Prof. E. Barberis for useful discussion on the interpretation of substrate analysis data. References Cordier, C., Trouvelot, A., Gianinazzi, S., Gianinazzi-Pearson, V., 1996. Arbuscular mycorrhiza technology applied to micropropagated Prunus avium and to protection against Phytophthora cinnamomi. Agronomie 16, 679–688.
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