The effect of growth substrate and fertilizer on the growth and vesicular-arbuscular mycorrhizal infection of three hosts MauritzVestberg Vestberg, M. 1992. The effect of growth substrate and fertilizer on the growth and vesicular-arbuscular mycorrhizal infection of three hosts. Agric. Sci. Finl. 1: 95-105. (Agric. Res. Centre of Finland, Laukaa Research and Healthy Plant Unit, Jun- nila, SF-41340 Laukaa, Finland.) The effect of growth substrate in combination with two compound fertilizers or bo- ne meal on the vesicular-arbuscular mycorrhizal (VAM) infection and growth of micropropagated strawberry (Fragaria x ananassa Duch.) and arctic bramble (Rubus arcticus L.), as well as maize (Zea mays L.) was studied in pot experiments. With all test plants, sand fertilized with bone meal was superior to the richer peat-based sub- strates in initiating rapid VA mycorrhizal infection and sporulation of the VAM fun- gi used. A fertilizing regime yielding plants of a sufficient size, which at the same ti- me are mycorrhizal, is suggested. Key words: Vesicular-arbuscular mycorrhiza, growth substrate, fertilizer, micropropa- gated strawberry, micropropagated arctic bramble, maize Introduction Most plants of economic value are naturally associated with vesicular-arbuscular (VA) fungi (Harley 1965). Strawberry is reported to benefit from VA mycorrhizae when the availability of phosphorus (P) is limited (Daft and Okusanya 1973, Holevas 1966), but also under standard cultivation conditions in fields with high levels of available soil P (Dunne and Fitter 1989, Werner et al 1990). The benefits of inoculating micropropa- gated or certified stock plants of strawberry are stressed by Kiernan et al (1984), Robertson et al. (1988) and Chavez and Ferrera—Cerrato (1990). In field experiments, maize showed great increases in shoot dry matter after inoculation with VAM (Khan 1972). Daft (1991) observed a near- ly 100 % growth increase of maize seedlings after VAM inoculation in sparce stands, but no dis- cernible effects in very dense stands. Daft and Okusanya (1973) found VAM inoculation es- pecially to increase the number of vascular bundles and also to stimulate pollen development in maize. Kothari et al. (1990), on the other hand, reported that VAM inoculation ofmaize had no influence on the shoot dry weight, but increased the area and dry weight of leaves by 30 %. Simultaneously they observed, however, a 16 % decrease ofroot dry weight and a3l % decrease of root length. No studies have been conducted on the VA mycorrhizae of arctic bramble. The most outstanding effect of VA mycorrhiza on plant growth is the enhanced phosphorus uptake in soils or growth substrates of low phosphorus availability (Mosse 1986, Cooper 1982, Fitter 1988, Dunne and Fitter 1989). However, vesicular-arbuscular fungi may thrive also in soils of high soil fertility (Gerdemann 1970), or in soils amended with P up to 160 mg/kg soil as was observed by Kiernan et al. in maize (1984). 95 Agric. Sei. Finl. 1 (1992) https://www.c-info.fi/en/info/?token=kSkGrVol8iWak-69.jndY9TREeyQuABqtY4Mgmw.AweTryZt_GaHXZ_CVlxaev1B_n8USB0A5sf8U19MxwAxaUtgtXPChRDyW0Yq9tNCW5Vslf-Ut0qwJjHgmnXkXKGgFVkwIQMZbqNtp72Eqf7UMcoNBMmJzqqg3ixmozZJou-XST9lepCPvTfBWBhpmt6W-tDzRKUXEIxum0RuRBrrCWEP70q-Gvr-NVSG7AxNW6ROao1Z2pmXaJvIJI0HKoHVz8dpytb05pV8UqdsGWwRunGdNKacRuAl154h9a5znJyQdAJHFQ Agric. Sei. Finl. 1 (1992) The aim of this investigation was to study the effect of VA mycorrhiza inoculation on strawberry, maize and arctic bramble growing on sandy or peaty substrates and fertilized with bone meal or mineral compound fertilizers. Further consider- ations include the nutrient level of a growth substrate required for adequate plant growth and mycorrhizal infection. All experiments were conducted as pot experiments in the glasshouse. Material and methods Experimental designs and practical performance To study the effect of substrate, fertilization and VAM inoculation on growth and VAM infection in three hosts, four pot experiments were carried out in the glasshouse. All plants were cultivated in plastic Vefi-pots (2.5 dl), which were placed on top of an approx. 2 cm layer of wet vermiculite. During the experiments, watering was done to the vermiculite substrate. Three experiments (nos 1, 3 and 4) were ofa split-plot design, while experi- ment 2 was a randomized block experiment. The number of replications varied from four to six. VAM fungi used, the duration of the experiments and the glasshouse minimum and maximum temperatures are shown in Table 1 . Growth substrates and fertilizers The basic growth substrates used in the experiments were fertilized light sphagnum peat (Vapo Bl or 82, Finland), unfertilized sphagnum peat (Vapo, Finland), vermiculite (3-V, Vermipu Finland) and sand. The content of N, P and K in peat Bl were 96, 72 and 144 mg/1 sub-strate, respectively, peat B 2 144, 108 and 216 mg/1, res- pectively, and in peat fertilized with a com- pound peat fertilizer (11N-11P-20K, Kemira) 55 mg, 55 mg and 100 mg/1 substrate, respectively. The basic substrates were used steam sterilized (three times on successive days, 1 h., 100 °C) or unsterilized. The amount of soluble minerals pre- sent in the growth substrates after steam steril- ization was not determined. All experiments had the same fertilizer treat- ments except experiment 4 in which no extra fertilizers were applied to the growth substrates. In one fertilizer (bone meal, Kemira), the phos- phorus was in a slowly soluble form. Pots given Table 1. Experiments conducted to establish the effect of substrate and fertilization on VAM infection and plant growth in three different crops. Number and aim of Plants VAM fungi Repii- Temperature, °C experiment used used cations Duration Min. Max. 1. Effect ofsubstrate Straw- Glomus 6 18 January 17.6 30.6 and fertilization berry mosseae - 27 March 1990 2. Effect of fertili- " " 25 January 18.2 30.6 zation - 18 April 1990 3. Effect of liming Maize " 4 29 January 18.2 31.0 and fertilization - 22 May 1990 4. Effect ofsubstrate Arctic G. Mosseae 5 10 January 15.5 35.7 bramble Glomus spp. - 24 July - isolate 2 1990 - isolate 3 - isolate 4 96 bone meal were also given a phosphorus-free Hewitt solution (Hewitt 1952) once a week, starting two weeks after onset of the experiment. Two compound mineral fertilizers, both at two concentrations, were also used in the experiments. "Puutarhan Y-2" (7N-SP-15K, Kemira) was a solid fertilizer, while "Puutarhan täyslannos" (14N-SP- -2IK, Kemira) was applied as a liquid fertilizer once a week. Table 2 shows the calculated amounts of P per liter of growth substrate at different fertilizer applications. Sand and unfertilized peat substrates were given dolomite lime (Saxo OY) 5 g/1 substrate. Plants Micropropagated plantlets of strawberry (Frågar ia x ananassa Duch.) 'Senga Sengana' were used as test plants in experiment 1 and 2. An Fl hybrid of maize (Zea mays L.) 'North star' was used in experiment 3 and in experiment 4 micro- propagated plantlets of arctic bramble (Rubus arcticus L.) 'Mespi' and 'Pima' were used. VAM inoculation The VAM fungi used were Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe supplied by the Rothamsted experimental station and Glomus spp. isolates nos 2,3 and 4 from our own collection. The inocula were produced by growing the VA mycorrhizal fungi for four months in maizeroots in sand cultures of low phosphorus contents. Each inoculum was a mixture of infected maize root pieces with adhering sand, hyphae and spores. Micropropagated plantlets of strawberry and pregerminated seeds of maize were inoculated with 1 ml of inoculum placed into their planting hole. A thin layer of the inoculum, 3.5 ml/pot, was placed in the middle of the pots where the arctic brambles were planted. Observations Growth response was evaluated by the following criteria: - Shoot dry weight - Percentage ofplant survival (arctic bramble) - Plant height (maize) Table 2. The amount of phosphorus in different growth substrates after applications of fertilizers. Amount of P, rng/l growth substrate 1. 2. 3. 4. 5. 6. Unfer- Bone Puutarhan Puutarhan Growth tili- meal Y 2 täyslannos Substrate zed + Hewitt 0.5 g 1.5 g 0.05% 0.1% A. Unsterilized sand - 79 25 75 25 50 B. Sterilized sand - 79 25 75 25 50 C. Peat (Bl)+vermiculite 58 137 83 133 83 108 + sand, sterilized (8:1:1) D. Raw peat+vermiculite 44 123 69 119 69 94 + sand, basic fertilized and sterilized (8:1:1) E. As D„ but (5:0:5) 27 106 52 102 52 77 F. Peat (B2)+vermiculite 86 165 111 161 111 136 + sand, unsterilized (8:1:1) G. Peat (B2)+vermiculite 86 165 111 161 111 136 + sand, sterilized (8:1:1) 97 Agric. Sei. Finl. 1 (1992) - Number of runners (strawberry) - Length of leaf stalk (strawberry) The percentage of infected root was determined (experiment 4) by the gridline intersect method (GiovAnnette and Mosse 1980). VAM infection was also estimated from the abundance of spores (experiments 1-4). A representative 50 ml root and soil sample from each pot was thoroughly washed in running tap water. The occurrence of spores was evaluated on a rating scale of 0-5 as follows: O = No infection 1 = Very poor infection, 1-5 spores/50 ml substrate 2 = Poor to moderate infection, approx. 5-20 spores/ 50 ml substrate 3 = Moderate infection, 20 - several hundred spores/ 50 ml substrate 4 = Abundant infection, spores abundantly 5 = Very abundant infection Results Experiment 1 Strawberries growing on unsterilized peat substrate (substrate F), where the content of phosphorus varied between 86 and 165 mg/1 substrate, had no VAM sporulation whatsoever after 68 days. When the same substrate was steam sterilized (substrate G), a few spores were observed in the unfertilized (86 P) and in the 7N-SP-15K (86 and 111 P) fertilized treatments. In unsterilized sand (substrate A), Glomus mosseae sporulated very abundantly when bone meal + Hewitt solution was used as fertilizer (79 P). Poor to moderate sporul- ation occurred in the unfertilized treatment. In treatments with the compound fertilizer 7N-SP- -15K at two concentrations (25 and 75 P), only a very poor sporulation was observed (Fig. 1). The shoot dry weight of strawberry correlated negatively with the occurrence of spores of G. mosseae. Strawberry grew best on average on the peat-based substrate F and poorest on the sand substrate A (Fig. 1). On unsterilized peat substrate the highest dry weights of strawberry, 2.98 and 3.00 g/plant, were achieved with bone meal + Hewitt solution and compound liquid fertilizer 15N-15P-21K, respectively. The dry weights were on sterilized peat, 2.01 and 1.59 g/plant, respect- ively. On unsterilized sand substrate, however, the liquid compound fertilizer, at concentrations of 0.05 % and 0.1 %, was superior to the other fertilizer treatments (Fig. 1 ). Experiment 2 When strawberry was growing on steam sterilized sand (substrate B) spores of G. mosseae were detected in all fertilizer treatments after 83 days. As in experiment 1, spores occurred most abundantly when bone meal + Hewitt solution were used as fertilizer (79 P). A moderate number ofspores were found also in unfertilized pots despite the ex- tremely poor growth in these pots. The com- pound fertilizer 7N-SP-15K (0.5 g/1 sand, 25 P) also gave a moderate number of spores, while in the rest of the treatments the occurrence was very low. The highest dry weights of strawberry were observed when 7N-SP-15K was used as fertilizer, 3.49 and 3.71 g/plant respectively, for the two concentrations. At a concentration of 0.5 %, good growth of strawberry and a moderate VAM infection were reported (Fig. 2). The number of runners per plant was highest at the highest dry weight: 3.8 and 4.0 for the compound fertilizer 7N-SP-15K (Table 3). In unfertilized control plants there were no runners at all. The length of the leaf stalk averaged 11.2 cm and 10.5 cm, respectively for the three largest leaves when 7N-SP-15K. (0.5 and 1.5 g/1) was used as fertilizer. Experiment 3 The level of dolomite lime applied had no significant effect on the appearance of Glomus mosseae spores in the pots with maize (Fig. 3) after 113 days. Similarly to experiments 1 and 2, spores appeared very abundantly in the treatment with bone meal + Hewitt (79 P). However, the dry 98 Agric. Sei. Finl. 1 (1992) Fig. 1. The effect of growth substrate and fertilization (see Table 2) onshoot dry weight and the occurrenceof spores (from - to +++++) around roots of micropropagated strawberry cv Senga Sengana inoculated with Glomus mosseae. 99 Agric. Sei. Finl. 1 (1992) Table 3. The effect of fertilization (see table 2) on the num- bers of runners and the average length of the three longest leaf stalks in micropropagated strawberry cv. Senga Senga- na inoculated with Glomus mosseae. Number of Height of Fertilization runners/plant leaf stalk, cm 1 Ö 3 2Ad 2 1.0“* 5.1 C 3 3.8” 11.2” 4 4.0a 10.5ab 5 1.5cb 9.6 b 6 9.2b Values in columns marked with the same letter do not differ at P=0,05 (Duncan) weight of maize plants in this treatment was low, varying between 2.62 and 4.34 g/plant after different liming treatments. In pots given the com- pound fertilizer 7N-SP-15K (25 and 75 P), in- fection was moderate to abundant, while the total dry weight of maize was considerably higher than in the bone meal + Hewitt treatment, varying from 1.87 to 12.04 g dry weight/plant. Maize growth was abundant after application of the liquid fer- tilizer 14N-SP-2IK (25 and 50 P), the total dry weight being 16.51 - 18.59 g/plant at the concen- tration 0.1 %. At the concentration 0.05 % liquid fertilizer a moderate to even abundant spore occurrence was noticed, while at the concentration 0.1 % spores did not occur or only very poorly. Experiment 4 Inoculation with VAM had on average a significant influence on total dry matter of arctic bramble. In 'Mespi', all the fungal strains used increased the total dry weight as compared to the control. The effect was greatest after inoculation with isolate 4. In 'Pima', isolate 3 gave the highest total dry weight (Table 5). Isolate 2 decreased the yield as compared to the control. Due to the spread of the introduced VAM fungi, almost all control plants of both 'Mespi' and 'Pima' exhibited root infection ( Fig . 4 ). In the sand sub- strate ( B ), control plants had a lower percen- tage of VAM-infection than in the peat-based substrates where plants showed a better growth. On average there were only minor differ- ences between the percentages ofVAM infection in roots of arctic bramble growing on different substrates with P-contents varying from very low (substrate B) up to 27 mg P/l (C), 44 mg P/l (B) and 58 mg P/l substrate. However, the highest infection rates occurred on the sand substrates both for 'Mespi' and 'Pima'. In contradiction to the percentage of root infection, the introduced VAM fungi sporulated moderately or abundantly only in the sand substrate (Fig. 4). The sporulation of strain 3, however, was neglictible or very poor also in this substrate. VAM-inoculation greatly Fig. 2. The effect of fertilization (see Table 2) on shoot dry weight and the occurrence of spores (from - to +++++) around roots of of micropropagated strawberry cv Senga Sengana inoculated with Glomus mosseae. Sand substrate. Bars marked with the same letter do not differ significantly at P=0.05. 100 Agric. Sei. Finl. 1 (1992) Agric. Sa. Fint. 1(1092) Fig. 3. The effect of dolomite lime application and fertilization (see Table 2) on shoot dry weight, final plant height and the occurrenceof spores (from - to +++++) around roots of maize cvNorth star inoculated with Glomus raosseae.Sand substrate. Bars marked with the sameletter do not differ significantly at P=0.05. 101 Fig. 4. The effect of growth substrate (see Table 2) and VAM inoculation (1, 2,3, 4,5: uninoculated, G. mosseaeandG. spp 3, 4 and 4, respectively) on total dry weight, VAM infection and the occurrence of spores (from -to +++++) around roots ofmicropropagated arctic bramble cvsMespi and Pima. Whole bar indicates total dry weight. Part ofbar marked with crosshatched lines indicates root dry weight. Bars marked with the same letter do not differ significantly at P=0.05. Num- bers below horizontal line indicate percentage ofroot infection. 102 Agne. Sei. Finl. 1 (1992) Table 4. The effect of vesicular-arbuscular mycorrhizal in- oculation on the percentage of surviving plants of arctic bramble cvs 'Mespi' and 'Pima' on a sand substrate after 195 days. % of surviving plants Inoculation ’Mespi’ ’Pima’ None 0 30 Glomus mosseae 50 70 Glomus sp., isolate 2 30 80 " " " 3 60 70 " " " 4 60 60 increased the percentage of surviving arctic brambles on the sand substrate (Table 4). Discussion Very abundant VAM sporulation was observed in experiments 1, 2 and 3 both in strawberry and maize in a sand substrate fertilized with bone meal, which gave a calculated P content of 79 mg slowly soluble P/l. The result is in agreement with other studies (Gerdemann 1968, Mosse 1973, Menge et al. 1978) according to which mycorrhizal infection best takes place under P-limiting con- ditions. Despite good mycorrhizal infection, the plants growing on a sand substrate fertilized with bone meal were always inferior in size to the plants given mineral fertilizers, which on average had only a poor to moderate VAM infection. A compromise must therefore be made in order to produce plants of sufficient size, which at the same time are mycorrhizal. In strawberry grown on a sand substrate, the compound fertilizer 7N-SP-15K used at a concentration of 0.5 g/1, giving a P content of 25 mg/1, seems to meet these re- quirements rather well (experiment 2). Maize achieved a moderate growth and VAM sporulation by applying 7N-SP-15K. at a concentration of 1.5 g/1 which gives a P content of 75 mg/1, or by applying the liquid fertilizer 14N-SP-21K. at a 0.05 % concentration weekly. Sporulation occurred also at fairly high levels of available substrate P. The highest phosphorus content, where sporulation of the introduced VAM fungi occurred, was observed at 111 mg P/l sub- strate for strawberry in experiment 1, which is in agreement with the findings by Gerdemann (1970) who claims that many crops in the Mid-West of the USA remained highly mycorrhizal despite high soil fertility. In experiment 3, sporulation of VAM was moderate in maize at a phosphorus level of 75 mg/1 soil. This observation supports the studies by Fairchild and Miller (1990) and Kothari et al. (1991) according to which VAM infection of maize was observed even after amend- ments with P of 100 mg/kg soil or more. On the peaty substrate in experiment 1, maxi- mum growth was achieved at a phosphorus Table 5. Significance of F-values in the analysis of variance for dry weight, plant height and mycorrhizal root infection of arctic bramble. Significance ofF-value Source Dry weight Plant Root ofvariation Shoot Root Total height infection A. Cultivar *** NS *** *** NS B. Substrate *** *** **� *** NS C. VAM NS ** NS *** A x B *»* *** *** *** NS A x C NS *** ** NS �* BxC NS *** ** NS ** A x B x C NS NS NS NS NS • P<0.05 ** P