The effect of inoculation with VA mycorrhizal fungi on the productivity of commercially grown strawberry, cv. Senga Sengana, was studied in a field experiment in ...
Plant and Soil 144: 133-142. 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.
PLSO 8757
Inoculation of commercially grown strawberry with VA mycorrhizal fungi M. NIEMI and M. VESTBERG Department of Biology, Espoo Research Centre, Kemira Oy, P.O. Box 44, SF-02271 Espoo, Finland and Agricultural Research Centre of Finland, Laukaa Research and Healthy Plant Unit, Juntula, SF-41340 Laukaa, Finland Received 30 May 1990. Revised March 1992
Key words:
field inoculation, micropropagated strawberry, pretransplant inoculation, VA mycorrhizae
Abstract
The effect of inoculation with VA mycorrhizal fungi on the productivity of commercially grown strawberry, cv. Senga Sengana, was studied in a field experiment in southern Finland. Micropropagated certified strawberry plants were inoculated at planting with different strains of Glomus spp. Although none of the inoculants raised the level of root infection above the natural infection level, all inoculated plants produced more runners in the first year than the control plants. Glomus intraradix Schenck & Smith (GI), G. etunicatum Becker & Gerdemann (GE) and Glomus sp. E3 (GF) significantly increased the number of runners by 57%, 69% and 76%, respectively. However, there was no significant increase in runner production in the second year, nor in fruit production in the third year. Of the strains tested, E3 was the most effective, increasing runner production by 30% over the first two years. Plants inoculated with G. mosseae (Nicol. & Gerd.) Gerdemann & Trappe (GM) produced fewer but larger runners than the control plants, and had a higher capacity for runner production relative to the plant size. The possibility of establishing mycorrhizal infection in micropropagated strawberries directly after the in-vitro phase under standard nursery conditions was studied in two glasshouse experiments. Three (GE, GF and GM) of five Glomus spp. caused mycorrhizal infection in plants of all four strawberry cultivars studied. In practical strawberry farming greater benefit of the mycorrhizal symbiosis may be achieved by using pretransplant-inoculated plants and adjusting the fertilizer regimes.
Introduction
Most of the important crop plants are naturally associated with vesicular-arbuscular (VA) mycorrhizal fungi, although their dependence on mycorrhizal symbiosis varies with plant species and environmental conditions. Strawberry, Fragaria × ananassa Duch, can benefit considerably from VA mycorrhizae when the availability of soil phosphorus (P) is limited (Daft and Okusanya, 1973; Holevas, 1966). Plenchette et al., (1982) found that VA mycorrhizal inocula-
tion of strawberry plants grown in calcined montmorillonite with standard fertilization increased the numbers of flowers and fruits, and sometimes also vegetative growth. A small benefit from mycorrhizae for strawberries with adequate nutrition was also recorded by Kiernan et al. (1984), who nevertheless concluded that the symbiosis was not essential for the growth of strawberry. In a recent study Dunne and Fitter (1989) obtained evidence that mycorrhizai fungi are beneficial to a strawberry crop growing under standard cultural conditions in a commercial field.
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They found that cultivated strawberries had an extremely high P demand and calculated P inflow rate later in the season and concluded that P uptake during reproductive development was assisted by mycorrhizal transport. Breeders have selected varieties with greatly enhanced fruit production and a consequently high P demand, which exceeds the capacity of the root system, at least during the reproductive phase (Dunne and Fitter, 1989). The presence of a functional symbiosis can thus be important, even under commercial cultural conditions with high fertilizer applications. Strawberry is routinely propagated from runners or by micropropagation. Initially it is grown in highly fertilized soilless mixes or fumigated soils, which allow little or no natural colonization of roots by VA mycorrhizal fungi. Consequently, the mycorrhizal status of certified strawberry nursery stock varies greatly (Robertson et al., 1988). Also strawberry production fields contain highly variable numbers of endomycorrhizal propagules, sometimes even less than 10 per kg soil (Robertson et al., 1988). Thus, subsequent colonization by indigenous mycorrhizal fungi is not guaranteed. VA mycorrhizal inoculation of strawberry plants prior to or at planting would ensure establishment of the symbiosis and possibly improve productivity, either by increasing the formation of runners (Holevas, 1966; Hrselovfi et al., 1988) or the numbers of flowers and fruits (Daft and Okusanya, 1973; Plenchette et al., 1982; Robertson et al., 1988). Biermann and Linderman (1983b) showed that both preand post-transplant inoculation increased the subsequent growth of geranium. An established and functioning symbiosis could also reduce the transplant stress observed with other transplanted crops (Menge et al., 1978). The present study assesses the benefit of VA mycorrhizal inoculation in practical strawberry farming. A field inoculation experiment was conducted with strawberry (cv. Senga Sengana) in a commercial field in southern Finland. Subsequently the possib?lity of obtaining mycorrhizal strawberry plants for outplanting by inoculating micropropagated plantlets directly after the invitro phase was studied in two glasshouse experiments with several VA mycorrhizal fungi and four different strawberry cultivars, grown under standard nursery conditions.
Methods Field experiment
The experiment was conducted in a commercial strawberry field located in southern Finland (N 60°07 ', E 23°35'). It was started in 1987 and the effect of VA mycorrhizal inoculation on strawberry growth and productivity was followed during the first three years of cultivation. Plants and inocula
Five-week-old micropropagated strawberry plants, cv. Senga Sengana, were obtained from the Laukaa Research and Healthy Plant Unit of the Agricultural Research Centre of Finland (Laukaa). As inoculants Glomus deserticola Trappe, Bloss & Menge (Rothamsted), G. etunicatum (Rothamsted), autoclaved G. etunicatum (Rothamsted), G. sp. E3 (Rothamsted), G. intraradix (NPI, Salt Lake City) G. mosseae (NPI, Salt Lake City), G. mosseae (UK, Rothamsted) and Nutri-Link (NPI, Salt Lake City) were used. Nutri-Link is a commercial granular spore formulation of G, intraradix. The other inocula were produced by growing the VA mycorrhizal fungi for four months in maize roots in sand-grit cultures. Each inoculum was a mixture of infected maize root pieces with adhering sand, hyphae and spores, and had a roughly estimated inoculum density of 60, 1600, 0, 500, 780 and 440 spores/ml for G. deserticola, G. etunicatum, Nutri-Link, G. mosseae (NPI) and G. mosseae (UK), respectively. The spore amounts of autoclaved G. etunicatum and G. intraradix was not estimated. The infection level of the maize roots in the inocula varied between 19 and 94% infected root length. All strains except Nutri-Link and G. mosseae (NPI) were used in the field experiment. Experimental design
The experimental design was a latin square with seven inoculation treatments and seven replicate blocks consisting of ten plants in a row. The experimental plot was surrounded by untreated plants, and the blocks were separated by one untreated plant to reduce cross-contamination via roots. The experimental plot was planted on June 16, 1987, and each plant was inoculated with 15 ml of inoculum placed in the planting
VA mycorrhizal inoculation of strawberry hole. Non-inoculated plants were used as controis (C). A second control treatment, intended to reveal possible physico-chemical effects of the inoculum carrier, was made by autoclaving a batch of GE. However, a subsequent viability test of the inoculants after one year of storage at +4°C revealed that 20 min autoclaving had not completely killed the fungus. The treatment GEa was thus the same as GE, but with a reduced infectivity of the inoculum.
Field site and cultural practices The field had not been cultivated for several years when in 1985 it was prepared for strawberry production by mechanical fallowing and basic fertilization (200 kg ha-~ P, 210 kg ha- ~ K plus trace elements, given as superphosphate, KC1 and a compound PK-trace element fertilizer). In 1986 the field was maintained free of weeds by glyphosate treatment. The soil was a silty clay with pH 5.9 and an ammonium acetate-extractable nutrient content in July 1986 of 1400 mg L-Z Ca, 185mgL -l Mg, 140mgL - 1 K a n d 14mgL l P as determined by standard soil analysis (Vuorinen and M~ikitie, 1955). In June 1987 the field was planted with a row distance of 140 cm and a plant distance of 33 cm. The soil in the rows was covered with black plastic. To enhance runner formation all flowers were removed in 1987 and 1988, and runners were collected between June and September. Both fruit and runners were harvested in 1989. The fertilizer regime was planned for runner production during the first two years and for fruit production in 1989. In 1987, the field was limed to pH6.5 with dolomite lime ( 6 t h a ~) and fertilized with 105kgha L N, 113kgha L p, 223kgha ~K, 180kgha ~S, 6 0 k g h a - 1 M g p l u s trace elements, given as superphosphate and a compound NPK-7-5-15-fertilizer. In April 1988, the field was fertilized with 49kgha -1 N, 3 4 k g h a ~ P, 104kgha l K, 85kgha-~ S, 2 8 k g h a -I Mg plus trace elements, given as a compound NPK-7-5-15-fertilizer, with an additional 100 kg ha J N given in June. In 1989 the applied amounts of N, P, K, S and Mg were 42, 29, 90, 73 and 24 kg ha L, respectively. In 1987, the areas between rows were herbicide-treated with simazin (Simatsin-neste, 2 L h a l) on July 10, and the foliage sprayed against grey mould with triadimephon (Bayleton
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25, 0.5 kgha ~) on July 20. Diquat herbicide (Reglone 0.5% and Agral 0.1%) was applied in September. Also in 1988 and 1989, an extensive plant protection programme was carried out with fungicides, herbicides and insecticides. Details are not given here, however, because root infection was studied only in the first year. The summer of 1987 was rainy, and irrigation was not needed. In 1988 and 1989, the field was irrigated twice a week (about 25 mm per week) during dry periods.
Sampling and measurements In 1987 plants were sampled on August 13, two months after inoculation. The fifth plant in each block was removed with part of the newly formed root system. It was later replaced with a new plant. Runners were counted, and the combined dry weight of shoots and runners was determined for each harvested plant. The relative runner production was calculated as the number of runners per g dry weight. A sample of newly formed roots was rinsed under running tap water and fixed in formalin:acetate:alcohol (1: 1:18) for subsequent estimation of the VA mycorrhizal infection. The percentage of infected root length was determined by the gridlineintersect method (Giovannetti and Mosse, 1980). At least 100 intersections of cleared roots, stained with 0.01% acid fuchsin in lactoglycerol were counted (Kormanik and McGraw, 1982). No attempt was made to distinguish between indigenous and introduced infection, In 1988, the seventh plant in each block was sampled before runner harvest, on July 21, and later replaced. In addition to counting the runners, the runner development stage was determined, using a scale of 1 to 4 (1 = primary runner plantlet developing, 2 = primary runner plantlet fully developed, 3 = secondary runner plantlet developing, 4 = secondary runner plantlet fully developed). The relative runner production was calculated as in 1987. The dry weights of runners and shoots were determined separately. The infection level of the roots was not determined. In 1989, the sampling was done on June 16, when fruit formation had begun but strawberries were not yet ripe. The above-ground parts of the third plant in each block were sampled. For each
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plant the numbers of flowers and fruits were recorded, and total dry weight of the aboveground phytomass was determined. Fruit formation relative to plant size was calculated as the total number of flowers and fruits per g plant dry weight. Runner formation had not yet started at the time of sampling.
periment ran from January 18 to February 17, and the plants were watered as needed, but not fertilized. Thirty days after inoculation, five replicate plants per treatment and cultivar were removed for shoot and root dry weight determinations. The VA mycorrhizal infection of three plants per treatment was estimated as described for the field experiment.
Glasshouse experiments
Experiment 2 Micropropagated, uniformly rooted plantlets of strawberry, cvs Senga Sengana and Hiku, obtained from the Laukaa Research and Healthy Plant Unit (Laukaa, Finland), were transplanted into 175-mL paper pots (type 608, Oy Potma Ltd, Finland). The potting mix consisted of eight parts horticultural peat (VAPO-B2, Finland), one part vermiculite (3-V, Vermipu, Finland) and one part non-sterilized sieved sand. The peat was a light Sphagnum peat, limed with dolomite lime to pH 6 and fertilized to give a final nutrient addition per litre potting mix of l l 5 m g N , 86 mg P and 173 mg K plus trace elements. The experiment was started on May 3, and five replicate plants per treatment were inoculated with 1 ml inoculum placed in the planting hole. The inoculants used were the same as in experiment 1 plus Glomus mosseae from Rothamsted (GMuk). They had been stored for 11 months at 4-8°C prior to use. The plants were transferred to a glasshouse with a temperature of 18-32/ 18°C day/night and no supplemental light, and kept during the first two weeks in a plastic tent covered with a fibre cloth to keep the relative humidity near 90% and the light intensity reduced. After 40 days the experiment was terminated, shoot and root dry weights were determined and VA mycorrhizal infection estimated as described above.
The possibility of obtaining mycorrhiza certified strawberries by pretransplant inoculation of micropropagated plantlets under commercial nursery conditions was studied in two glasshouse experiments. Experiment 1 Micropropagated, rooted plantlets of strawberry (cvs. Senga Sengana, Jonsok and Zefyr) were obtained from a commercial micropropagation laboratory (Hortus Oy, Kaarina, Finland) and grown under standard cultural conditions. The potting mix consisted of three parts horticultural peat and two parts ground rockwool (Grodan). The peat was a light Sphagnum peat (ST-2, Satoturve, Finland), limed with dolomite lime to pH 6 and fertilized to give a final nutrient addition per litre potting mix of 66 mg N, 74 mg P, 106 mg K, 33 mg S plus trace elements. In-vitro plantlets were transplanted into growing trays with 20 mL cells (Veil A/S Norway), filled with non-sterilized potting mix and inoculated with Glomus deserticola (GD), G. etunicatum (GE), G. sp. E3 (GF), G. intraradix (GI), G. intraradix 'Nutri-Link' (Glnl) and G. mosseae (GMnpi). The inoculants (see field experiment for details) had been stored at 4°C for six months, since the field experiment. The inoculum was mixed into the potting mix at a rate of 1 mL per plant, with ten replicates per inoculation treatment and cultivar, and with ten noninoculated plants as controls (C). The plants were acclimatized in a growth chamber with 23°C, 90% relative humidity and 16h/day with 2000 lux (Osram Fluora-Lux: Daylight 5000 DeLuxe 1:1). After two weeks the plants were transferred to a glasshouse with 22/18°C day/ night, 16h/day 15000 lux (Na-high pressure lamps) and 60-70% relative humidity. The ex-
Statistical analysis Data from the field experiments were statistically analysed with analysis of variance for the latin square design, and glasshouse data with one-way analysis of variance. When inoculation effects proved significant at p = 0.05, treatment means were separated by Duncan's test or the SNK test.
VA mycorrhizal inoculation of strawberry Results
Field experiment Two months after planting, the newly formed roots were well colonized by VAM fungi in all treatments, including the control (Table 1). Although the soil had been fallowed for two years, it still contained enough propagules of mycorrhizal fungi for a natural infection level of 14% to develop in the non-inoculated plants. The introduced infection could not be distinguished from the indigenous infection, and inoculation did not raise the infection above the natural level. There were no significant differences in dry matter between treatments (Table 1), although all inoculants except GMuk tended to increase plant dry weight. At the time of sampling, the inoculants GE, GF and GI had significantly increased the absolute number of runners per plant by 57, 69 and 76%, respectively (Fig. IA). The relative runner production was calculated to demonstrate the capacity for runner formation relative to plant size, which varied both within and between treatments. The autoclaved GE (GEa) and GD treatments did not differ from the control, while all other inoculants significantly increased the relative runner formation by 41-55% (Fig. 1B). Despite the smaller plant size and lower absolute number of runners, the relative runner production of GMuk-inoculated strawberries was as high as with the other inoculants and higher than that of the control plants of the same size.
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By July 1988, the plants had produced 3-6 times more runners than by August the year before, but there were no significant differences between treatments in absolute or in relative runner production (data not shown). GE had increased the number of runners by 26%, but due to the large variation between replicate plants in the field, this increase was not significant. None of the inoculants significantly increased the shoot or runner dry weight, nor was runner development faster (Table 1). However, plants inoculated with GMuk produced significantly larger runners than the uninoculated plants. In 1989, sampling occurred during flowering and early fruit formation, and the combined number of flowers and fruit was considered an estimate of the fruit yield. Again neither the absolute nor the relative fruit yield differed significantly between treatments (Fig. 2).
Glasshouse experiments When micropropagated plantlets of four strawberry cultivars were inoculated prior to transplant into peat-based potting mixes after the in-vitro phase, few differences in plant dry weight were observed (Table 2). In experiment 1, there were no differences in shoot and root dry weights for any cultivar. In the second experiment, the only significant inoculation effect was the increased root weight of GMuk-inoculated Senga Sengana plants (Table 2). The level of VA-mycorrhizal infection in the pot experi-
Table 1. VA mycorrhizal infection in the first year and growth in the first, second and third year of field-grown strawberry, cv. Senga Sengana, inoculated at planting with VA mycorrhizal fungi Inoculation treatment
C GD GE GEa GF GI GMuk
August 1987
July 1988
June 1989
lnf root length ( % )
Total D M ~ (g/plant)
Shoot DM (g/plant)
Runner DM (g/plant)
Mean runner dry weight (g)
Runner development stage
Total D M ~ (g/plant)
14.4a ~ 14.5a 10.2a 13.6a 10.4a 15.5a 12.3a
8.6a 9.7a 9.8a 10.8a 10.7a 9.9a 8.3a
101.8a 96.8a 97.3a 96. la 97.3a 91.2a 87.3a
8.6a 9.9a 15.2a 12.5a 10.2a 13.6a 14.7a
0.30a 0.33ab 0.42ab 0.40ab 0.34ab 0.47ab 0.64b
2.7a 2.9a 3. la 2.6a 2.7a 3.0a 3.0a
39.0a 31.8a 35.5a 28.9a 31.9a 28.4a 32.0a
A b o v e - g r o u n d phytomass. ~' M e a n s followed by the same letter within columns do not differ significantly at p - (I.05.
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