Effect of inoculation with arbuscular mycorrhizas on rooting, weaning and subsequent growth of micropropagated Malus (L.) Moench Marjatta Uosukainen and MauritzVestberg Uosukainen, M, & Vestberg, M, 1994, Effect of inoculation with arbuscular mycorrhizas on rooting, weaning and subsequent growth of micropropagated Malus (L.) Moench. Agricultural Science in Finland 3: 269-279. (Agricultural Research Centre of Finland, Laukaa Research and Elite Plant Unit, FIN-41340 Laukaa, Finland.) The importance of different rooting methods and the effects of arbuscular my- corrhizal (AMF) inoculation on the rooting rate, weaning survival and subsequent growth of microcuttings of Malus rootstock YP and the Finnish crab apple cultivars Hanna and Marjatta were studied in four experiments conducted at the Laukaa Research and Elite Plant Unit, Finland. Sucrose in the in vitro rooting medium gave the best support to weaning survival in comparision with glucose and fruc- tose. Directly rooted microcuttings had a higher survival rate than in vitro rooted microcuttings. AMF inoculation did not improve the rooting rate in direct rooting. However, the Glomus hoi strain V9B caused severe rotting of microcuttings and thus lowered the rooting rate of cv. Hanna, AMF inoculation increased the mean shoot height of established plants, particularly inoculation with G. hoi V9B, G. claroideum V43a or G. flstulosum Vl2B. This effect, however, varied considera- bly in different host-fungus combinations. Some AMF strains, e.g. G. hoi VIO4, caused strong growth retardation. After the rooting and weaning stage, many unin- oculated plants lapsed into arrest of growth. This phenomenon was less frequent in AMF inoculated plants. Optimal timing of AMF inoculation and nutritional level of rooting and weaning substrate are discussed. Key words: Apple rootstock, crab apple, carbon drain. Glomus claroideum, G. fistulosum, G. hoi, G. mosseae, rooting of microcuttings Introduction The beneficial effect of arbuscular mycorrhizal fungi (AMF) on the growth of plants is well doc- umented (Mosse 1973, Harley and Smith 1983). Apple seedlings are usually reported to show a strong positive growth response to inoculation with AMF (Benson and Covey 1976, Hoepfner et al. 1983, Gnekow and Marschner 1989, An et al. 1993), even at moderate or high phospho- rus availability (Plenchette et al. 1981, Gne- kow and Marschner 1989). In micropropagated apple plants AMF may be even more beneficial than in seedlings (Gianinazzi et al. 1990, Bran- zanti et al. 1992), although there are problems such as high sensitivity of apple microcuttings to rot following inoculation, particularly in direct rooting (Avanzato et al. 1994). The basic principles in rooting of apple micro- cuttings are rooting in vitro on agar gelified me- dium or direct, post vitro rooting on artificial or compost substrate (Dirr and Heuser 1987). Also combining of these two rooting methods was shown to be successful on rooting of apple mi- crocuttings (Zimmerman and Fordham 1985). In rooting of apple microcuttings, there may be large 269 Agricultural Science in Finland 3 (1994) https://www.c-info.fi/en/info/?token=IyILVF5ZROC8I3nE.cZaotG_Fq5LQZ1jOgHzmlg.R-kb9d01cFAsIxUeB7o5EDQdc6YLxiq9RRtTHjq1T7dzmzM65_K6WyYaecs0nre-FRyFI9D3tlzQ7mQf9hUp-kISqdvflNaZJqyj9zSQwiMyKkZLDR8HVUV5LUF8pfbwxe49VOrC4ynJkPv6LWt4fUvKJkl-VKxB4Z-WMWfrfzAY8JWh9CdL30sQiepMD4A-gdqEWPleKUdpS-6IUyFAZK2n4sZtF_4vWX4ShDdASeZU5PVrvrL4vaKiD0vQgDF4HFfbLmz_8Hp9ObB7g2uFv5NdY8B5vQK6pY9m_ovC fluctuations due to various accidental external fac- tors (Denissen et al. 1992). The timing of har- vesting shoots from the subculture medium (De Klerk and Ter Brugge 1992, Uosukainen 1992) and the type of carbohydrate used in a gelified rooting medium (Moncousin et al. 1992, Uosu- kainen 1992) are crucial. A too long auxin treat- ment may lead to rooting inhibition and distur- bances in the apical growth of micropropagated shoots (George and Sherrington 1988). Also light, photoperiod, temperature and macronutri- ents of the rooting medium have an effect on root initiation, root growth and the quality of the root system (Zimmerman and Fordham 1985, George and Sherrington 1988). A good rooting result in the laboratory does not always lead to good weaning and undisturbed continuous growth of the rooted plantlets. There may be great losses after transplanting to soil in greenhouses or in growth chambers (Preece and Sutter 1991). Microcuttings, rooted on agar, have difficulty in inducing a fully effective root sys- tem when the plants are transferred to soil (De- bergh and Maene 1981, Georgeand Sherrington 1988,Preece and Sutter 1991, Uosukainen 1992). During the rooting process, problems may arise also from internal latent bacterial contaminants (Boxus andTERZi 1985, 1988, Constantine 1986, Yeoman 1986, Poulsen 1988, Leifert et al. 1991, Savela and Uosukainen 1994). Also the quality and texture of the growth substrate and the ther- mohydrical stress influence the rooting and surviv- al of rooted plantlets, particularly in direct rooting (Avanzato et al. 1993). Good rooting techniques are needed to obtain plant material of uniform quality which, together with correct timing of AMF inoculation, are es- sential factors in AMF utilization. The aim of this study was to optimize these two steps and to minimize the effect of accidental external factors in AMF utilization. Material and methods The study consisted of four experiments. Apple rootstock YP, and two Finnish crab apple culti- vars, Hanna and Marjatta, were used in this study. All these cultivars are offspring of Malus hacca- ta (L.) Moench. Both ’Hanna’ and ’Marjatta’ cultures consisted of one meristem clone, but in the YP experiments, two meristem clones (YP- -130 and YP-148) were used. Microcuttings were 26-35 mm long and harvested from a prolifera- tion medium (Uosukainen 1992) six weeks after subculture. In the first three experiments micro- cuttings were rooted in test tubes, size 22 mm x 150 mm, sealed with aluminium caps allowing gas exchange. An acute auxin treatment (Lane and McDougald 1982) for root induction was given by immersing the microcuttings in 100 mg/ lIBA 1:1 alcohol-water solution for 15 min. Dur- ing the root initiation phase microcuttings were on the hormone-free rooting medium (Uosukai- nen 1992). Rooted microcuttings were transplant- ed to soil after 7 weeks. In experiment 4, the direct rooting method, combined with a proce- dure of acute auxin treatment, was used. Shoots were dipped for 7 sec in a water solution of NaOH (0.5N) dissolved IBA (100 mg/1). After dipping the microcuttings were stuck directly in rooting substrate. The AMF strains used as inoculants were Glomus claroideum Schenck & Smith strain V43a (experiment 2), G. hoi Berch & Trappe strains V9B (exps. 1 and 4) and VIO4 (exps. 1 and 2), G. mosseae (Nicol. & Gerd.) Gerdemann & Trappe strain V57 (exp. 4) and G. fistulosum Skou & Jakobsen strains V92 (exp. 1), VIO7 (exp. I) and VI2B (exp. 4). The inoculum was a mixture of infected maize roots, hyphae, spores and growth substrate. As a control, a similar soil and root inoculum without AMF was used. Inocula (1 ml/ microcutting) were placed in the planting hole prior to transplantation. Root colonization by AM fungi was estimated at the end of the experiment 1 and at the end of the first repetition of the experiment 4 using the grid-line intersect method (Giovannetti and Mosse 1980) for root samples stained in 0.05 acid fucsin according to Kormanik and McGraw (1982). Statistical analyses were done using SAS, and means were separated by using the Duncan’s Multiple Range Test. 270 Agricultural Science in Finland 3 (1994) In YP experiment I, the effects of fructose, glucose and sucrose (30 g/1), as carbohydrate source in the in vitro rooting medium, combined with AMF inoculation at transplanting to soil, on the survival and growth of 76 micropropagated YP plants were studied in a pot experiment with seven replicates. There were five mycorrhizal treatments: (I) uninoculated (No AMF), (2) inoc- ulated with V92, (3) inoculated with VIO7, (4) inoculated with V9B and (5) inoculated with VIO4. Rooted, 7-week-old microcuttings were planted in 0.25 1 plastic Vefi pots containing a substrate mixture of steam sterilized peat and sand (3:1) limed with 4 g/1 Dolomite lime (Saxo oy, Fin- land) to give an approximate pH of 6, and ferti- lized with 1.8 g/1 of the controlled-release ferti- lizer Osmocote Plus (8-9 months, 18N:11P:10K, Sierra Ltd, UK). The plants were transplanted to soil and inoculated on September 2, 1991. Plants were kept in continuous growth in a greenhouse for 34 weeks in long day. From October to March, plants were given artificial light (Philips HPI 400 W). The daily temperature was 18°C during the light photoperiod and 15°C in the dark. The ex- periment was completed on April 24, 1992. Shoot height was recorded at 18 weeks, 24 weeks and 34 weeks after inoculation. In YP experiment 2, the effect of AMF inocu- lation on the subsequent growth during the first two growing seasons of in vitro rooted YP mi- crocuttings was studied in a pot experiment with 14 replicates. Rooted YP microcuttings, altogether 78 plants, originated from a rooting medium con- taining sucrose 30 g/1. There were four AMF treat- mens: (1) uninoculated, (2) inoculated with V43a. (3) inoculated with VIO4 and (4) inoculated with a mixture of V43a and VIO4. Rooted microcut- tings were planted in 0.25 1 plastic Vefi pots con- taining a substrate mixture of steam sterilized peat, sandand finesand (1:1:1). The mixture was limed with 3 g/1 Dolomite lime (pH of the mixture about 6) and fertilized with 0.5 g/1 of Osmocote Plus (8-9 months). The rooted plantlets were transplanted to soil and inoculated on January 9, 1992. Plants were in continuous growth in the greenhouse until May 1992, The plants were kept in long day under artificial light (Philips SON-T Agro 400 W) from January to March 1992. The daily temperature was 18°C during the light photoperiod and 15°C in the dark. In May 1992, the plants were re- planted in 0.5 1 black plastic pots and kept out- doors from May to September 1992. At the be- ginning of October 1992, the plants were replant- ed in 2 1 plastic bag containers and stored at 4°C in the dark until the beginning of May 1993. Af- ter the cold storage, the plants were kept out- doors until October 11. 1993, whereafter the ex- periment was finished. During the first growing season, shoot heights were recorded 9 weeks af- ter transplanting, on the second growing season, immediately after winter storage and again at the end of the season, 76 and 91 weeks after trans- planting and inoculation, respectively. Also shoot base diameter was measured after 76 and 91 weeks, respectively. In YP experiment 3, the optimal timing for transplanting microcuttings to soil was studied on apple rootstock YP. Rooting and survival were compared between microcuttings kept on hor- mone-free in vitro rooting medium for 0 (=direct rooting), 2,3, 4 or 5 weeks before transplanting to soil. The experiment was repeated four times between June 1992 and January 1993. The root- ing and weaning survival of 480-500 shoots in each treatment, altogether 2457 shoots, was esti- mated. The rooting rate was recorded before trans- planting. Survival rates were estimated after two months in the greenhouse. Also pH of the rooting medium was measured during the in vitro rooting stage. In crab apple experiment 4, the effect of AMF inoculation on the rooting rate of crab apple cul- tivars Hanna and Marjatta was studied by using direct rooting with simultaneous AMF inocula- tion with three different AMF and a control inoc- ulum. Inoculations with control (No AMF) and AMF Vl2B were repeated four times. Inocula- tions with AMF V57 and AMF V9B were repeat- ed 3 and 2 times, respectively. Repetitions were started on February 18th, on April 20, on July 5 and on November 4 in 1993. Altogether 334 mi- crocuttings of cv. Marjatta and 241 microcuttings of cv. Hanna were stuck. Steam sterilized rooting 271 Agricultural Science in Finland 3 (1994) substrate, peat:sand:perlite (4:1:5), consisted of peat (Vapo Peat XL/80, Finland), sand (local, 0.5-1.5 mm) and perlite (coarse, 3-5 mm, Nord- isk Perlite, Denmark). The substrate was ferti- lized with 1 g/1 Osmocote Plus (8-9 months) and Dolomite lime 5 g/1. Microcuttings were rooted in plant propagators (WARD Seed Tray 355 mm x 215 mm with Seed Tray Cover Ward Reg. Des. N:o 938270), one treatment/tray during rooting and weaning. Trays were placed for four weeks into a growth chamber. The light source consist- ed of a combination of fluorescent lamps (Warm White Super and Floralux), 3500 lux-3700 lux inside the propagators. The temperature during the rooting and weaning periods was 20°C dur- ing a 16-hour light period and 16-18°C during the 8-hour dark period. Rooting and weaning were estimated after four weeks. After the rooting and weaning stage, subse- quent growth of the microcuttings, which were transplanted to soil and inoculated with G. fistu- losum VI2B on February 18th, was also estimat- ed. Two months after transplanting and inocula- tion, plant heights were measured, small plants were replanted in plastic pots (12 cm) and potted plants were placed in a randomized order on a table in the greenhouse. The potting substrate was similar to the rooting substrate. Plant heights were measured on May 6th, 2.7 months after inocula- tion, again on June 24th, during the most active growth, and finally in the autumn, on September 15th, 7 months after transplanting to soil. Results In YP experiment 1, weaning survival of apple rootstock YP was highly dependent on the carbo- hydrate source of the in vitro rooting medium. After 18 weeks in the greenhouse, the weaning survival rate was 0%, 21.1% and 50% for fruc- tose, glucose and sucrose, respectively. Due to the poor survival rates, the interactions between the carbohydrates and the mycorrhizal fungi were not recorded after the weaning stage. AMF inoculation increased the mean shoot heights, especially at 24 and 34 weeks. This effect Shoot height, cm was, however, not statistically significant (Fig. 1). Plants inoculated with G. hoi V9B resulted eventu- ally in 3 times higher plants than uninoculated ones. Also a higher rate of uninoculated plants lapsed into arrest of growth than inoculated plants, even- tually (Table 1).At 18 weeks, 22% of uninoculated and 18 %of inoculated plants were growing vigor- ously, being >5 cm high. At 24 weeks, 12.5% of uninoculated and 22% of inoculated plants were >lO cm high, at 34 weeks 12.5% and 55.5%, re- spectively. At the end of the experiment, the AMF colonization in inoculated plants varied from 25% (V92) to 37.5% (V9B) . Due to spread of AMF from adjacent pots, AMF colonization was 6.3% in the uninoculated plants. Fig. 1. Effect of arbuscular mycorrhizal (AMF) inocula- tion (V92, VlO7 = G. fistulosum\ V9B, VlO4 = G.hoi) on shoot height of apple rootsock YP at three different times. For each time, values sharing the same letter do not differ at p=0.05 according to Duncan's multiple range test. No AMF = non-inoculated control plants. 272 Agricultural Science in Finland 3 (1994) Table I. Effect of arbuscular mycorrhizal (AMF) inocula- tion (V92, VIO7 = G.fistulosum; VIO4 =G. hoi) on the proportion ( %, as means of 4-9 surviving plants) of ap- ple rootstock YP plants in vigorous growth (i.e. plants >lOO mm) in height, at 24 weeks and 34 weeks after onset of pot experiment. No AMF = non-inoculated control plants. Plants in vigorous growth, % Root Mycorrhizal colonization, % treatment 18 weeks 24 weeks 34 weeks 34 weeks NoAMF 22.2 12.5 12.5 6.3 b V92 0 25.0 25.0 25.0ba VIO7 25.0 0 40.0 30.0 a V9B 33.3 20,0 100.0 37.5 a VIO4 14.3 42.9 57.1 26.4ba Inoculated, mean 18.2 22,0 55.5 29.7 The results from the YP experiment 2 are il- lustrated in Figure 2. After nine weeks, there was a significant growth increase in YP root- stocks due to inoculation with G. claroideum V43a. The other AMF strains also caused a mean growth increase, which was, however, not statis- tically significant. At 76 weeks, after cold stor- age and one month of new growth, the positive effects of inoculation had decreased. However, plants in the treament V43a + VIO4 were, on average, still bigger than plants in other treat- ments, but the differences were not statistically significant. Prior to cold storage, this treatment had the shortest plants. Plants inoculated with VlO4 alone were poor in growth after cold stor- age. There were only small differences in the stem base diameterbetween inoculated and unin- oculated plants. After cold storage, VIO4 showed also the smallest stem base diameter which cor- relates with the poor growth in this treatment. The uninoculated treatment had the highest pro- portion of surviving plants throughout the exper- iment, the percentage being 86% at the end of the experiment. Corresponding percentages for the AMF treatments V43a, VlO4 and V43a+Vlo4 were 57%, 29% and 64%, respectively. Root infection was not evaluated in this experiment. At 9 weeks, the proportion of plants in active growth (>IO cm high) was greater in all AMF treatments (Table 2) than in the control. At 76 and 91 weeks, however, inoculation with G. hoi VlO4 had caused a strong growth retardation, while 100% of the plants inoculated with the com- bination of V43 and VlO4 were >3O cm high, eventually. In YP experiment 3, the roots started to emerge after 10 days on the in vitro rooting medium. The mean rooting rate of microcuttings at transplant- ing and the mean weaning survival of the same plantlets after 2 months in the greenhouse showed negative correlation (Fig. 3). Microcuttings that had been on the in vitro rooting medium for 5 weeks had the highest mean rooting rate, 41%, but the mean survival rate was only 27%. Direct- ly rooted microcuttings had the highest mean sur- vival rate, 73.3%. Although there was seasonal Fig. 2. Effect of arbuscular mycorrhizal (AMF) inocula- tion (V43a = G. claroideum , VlO4 = G. hoi) on shoot height, proportion of surviving plants and stem base di- ameter of apple rootstock YP at three different times. For each time, values sharing the same letter do not differ at p=0.05 according to Duncan's multiple range test. No AMF = non-inoculated control plants. 273 Agricultural Science in Finland 3 (1994) variation in percentual rates between repetitions, the rooting rates correlated with the survival rates. In visual observation, an increasing number of symptoms resembling nutritional deficiency was recorded in the microcuttings during the in vitro Table 2. Effect of arbuscular mycorrhizal (AMF) inocula- tion (V43a =G. claroideum; VIO4 =G. hoi) on the pro- portions (%, as means of 4-12 surviving plants) of apple rootstock YP plants in vigorous growth, i.e. >IOO mm high at 9 weeks, >250 mm at 76 weeks and >3OO mm at 91 weeks after onset of pot experiment. No AMF = non- inoculated control plants. Plants in vigorous growth, % AMF treatment 9 weeks 76 weeks 91 weeks No AMF 0.0 41.7 58.3 V43a 37.5 37.5 75.0 VIO4 28.6 0.0 25.0 V43+V104 22.2 77.7 100.0 rooting. The pH value of the in vitro rooting me- dium rose during the 5- week period from 4.9 to 6.3. According to the results obtained from crab apple experiment 4, AMF inoculation did not im- prove the rooting rate in direct rooting (Table 3). Malus cultivars differed in their reaction to dif- ferent AMF. Particularly AMF V9B seemed to be incompatible with cv. Hanna. In visual observa- tion, at the end of the rooting stage, the uninocu- lated plantlets seemed to be more vital than the inoculated ones. Most of the inoculated plants had dry or pale green leaves often with a tinge of red. These symptoms were transient in their na- Fig. 3. Effect of length of in vitro rooting phase (wk) on rooting and survival rates ( %) of apple rootstock YP mi- crocuttings. Rooting rates were estimated at transplanting to soil and survival rates 2 months after transplanting. Fig. 4. Effect of arbuscular mycorrhizal (AMF) inocula- tion (VI2B =G. fistulosum) on the continuous growth of directly rooted microcuttings of crab apple cultivars Mar- jatta and Hanna. No AMF = non- inoculated control plants. The proportion of growing plants ( %) after the rooting and weaning stage was estimated on May 6, June 24 and September 9 in 1993. 274 Agricultural Science in Finland 3 (1994) lure and resembled symptoms caused by nutri- tional deficiencies. The plantlets, inoculated with G. fistulosum VI2B, continued their growth and development soon after the weaning stage. The uninoculated plants, instead, had a great tendency to lapse into Table 3. Effect of arbuscular mycorrhizal (AMF) inocula- tion on rooting rate (%) of direct rooted microcuttings of crabapple cultivars Marjatta and Hanna, recorded four weeks after inoculation and transplanting to soil. Inocula- tion treatments (AMF V57 = G. mosseae; AMF V9B = G. hoi; AMF VI2B =G. fistulosum and No AMF =no inocu- lation, given to coincide with transplanting to soil), total number of treated microcuttings and rooting rate ( %, as means of4 repetitions). Cultivar Treatment Number of Rooting microcuttings rate, % Marjatta No AMF Marjatta AMF V57 Marjatta AMF V9B Marjatta AMF VI2B Hanna No AMF Hanna AMF V57 Hanna AMF V9B Hanna AMF VI2B 103 77.7 73 80.8 69.058 100 65.0 75 66.7 55 63.6 30.040 71 77.5 arrest of growth (Fig. 4). The greatest difference was observed 2.7 months after inoculation and transplanting to soil. At that point, on May 6th, approximately 90% of inoculated plants had start- ed active growth. However, at the same time only about 60% of uninoculated plants were in active growth. Even 4 months after transplanting and inoculation, only 58.3% of uninoculated 'Hanna' plants had started to grow in height. After 7 months, on September 15th, 8.3% of uninoculat- ed 'Hanna' plants had remained at the stage of dormancy. The growth curves of directly rooted and G. fistulosum Vl2B inoculated 'Marjatta' and 'Han- na' plants (Fig. 5) illustrate that the inoculated plants were, throughout the growing season, at least 19% higher than the uninoculated plants. However, the difference in growth was statisti- cally significant only in cv. Hanna, on May 6th, 2.7 months after transplanting to soil. At that point the inoculated 'Hanna' plants were 76% higher than the uninoculated plants. The major differ- ences in growth decreased towards the end of the growing season. Only sporadic AMF root coloni- zation was observed after 4 or 7 months despite a Fig. 5. Effect of arbuscular mycorrhizal (AMF) inoculation (Vl2B = G. fistulosum) on the growth of micropropagated crab apple cultivars Marjatta and Hanna. No AMF = non-inoculated control plants. The plant height (mm) was measured on April 15, May 6, June 24 and September 15. 275 Agricultural Science in Finland 3 (1994) moderate to abundant sporulation in cv. Marjatta and a moderate sporulation in cv. Hanna. Discussion The experiments conducted with apple rootstock YP and crab apple cultivars Hanna and Marjatta show both a negative and a positive effect from inoculation of AMF fungi at the beginning of the weaning stage of the microcuttings. Of the posi- tive effects, increased growth as a result of inoc- ulation with Glomus claroideum V43a, G. fistu- losum Vl2B and G. hoi V9B can be mentioned. Most of the AMF inoculated plantlets obtained continuous normal growth soon after the wean- ing stage, but growth lapses of several months were common in uninoculated plantlets. There- fore, the inoculated plants were more uniform at the end of the growing season. From a practical point of view, this means a great benefit for the nursery: due to the balanced growth, the majority of the plantlets can be sized into one class. AMF inoculation can be profitable to nurseries also due to the shorter time needed for raising the plant- lets to saleable trees. According to our results, the culture time may be a quarter to one third shorter than with uninoculated plants. In apple, the benefits of early inoculation of micropropagated plantlets should be re-evaluat- ed. In direct rooting of microcuttings, AMF inoc- ulation did not give such a positive effect on rooting as has been reported on bigger cuttings (Johnson et al. 1980, Gnekow and Marschner 1989). On the contrary, G. hoi V9B seemed to be incompatible with crab apple ’Hanna’, causing severe rotting of the microcuttings. A similar neg- ative effect of AMF inoculation on Malus micro- cuttings in direct rooting was also observed by Avanzato et al. (1994) using G. mosseae as the inoculant. The poor general condition of the in- oculated microcuttings after the rooting and wean- ing stage, possibly due to nutritional deficien- cies, may be caused by too rapid and intense root colonization by the AM fungi. AMF inoculation of in vitro rooted plantlets simultaneously with transplanting to soil did not seem to be beneficial, either. As a rule, in on agar rooted microcuttings, most AMF-host combina- tions resulted in lower survival rates during plant establishment to soil than no inoculation. Strains belonging to the species G. hoi, in particular, seemed to cause severe rotting of the plantlets. In the present study, the rotting of microcuttings and micropropagated plantlets was probably not caused by a pathogen, because (i) the control plants were inoculated with the same soil inocu- lum but withoutAMF, (ii) some AMF strains gave better rooting results than others and caused only little or no rotting, (iii), rotting occurred slowly, after three weeks. Thus the rotting did not start until the roots had emerged and root growth had started. Bacterial contaminants that cause rotting of microcuttings (Boxus and Terzi 1985, 1988, Constantine 1986, Yeoman 1986,Poulsen 1988, Leifert et al. 1991, Savela and Uosukainen 1994) appear virulent earlier and are more acute in their nature. Apple microcuttings need about 4 weeks to develope a good root system and they seem to change to fully autotrophic nutrition slow- ly. During this period plantlets normally release more CO, than they are capable to assimilate through photosynthesis (George and Sherrington 1988). It is therefore concluded that the rotting of the microcuttings was actually caused by the mycorrhizal fungi. Infection with AMF during the early acclimatization phase probably led to a carbon drain which was not compensated for by the plant during the prevailing stress conditions. Carbon drain is a phenomenon not uncommon in mycorrhizal experiments. Gnekow and Marschn- er (1989) suggested that carbon drain takes place in AMF inoculated apple cuttings in a field situa- tion at high P levels. It is a well-known fact that Malus microcut- tings are extremely sensitive to environmental stresses (Dirr and Heuser 1987,Avanzato et al. 1993). The adaptation to post vitro conditions, as such, is an extreme stress to a small plantlet due to a high energy requirement. Therefore, AMF inoculation may become an overwhelming stress for a small apple microcutting at the rooting and weaning stage. If the small plantlet, often in poor nutritional and hormonal balance, is sub- 276 Agricultural Science in Finland 3 (1994) mitted simultaneously to another extreme stress, such as AMF infection, the result may be a host- parasite relationship instead of a symbiosis. According to Avanzato et al. (1994), the rot- ting might have been due to some pathogen in the inoculum. To overcome the rotting problem, he suggests that the inoculum should be applied as sterilized spores. In France, Ravolanirina et al. (1989) have developed a method of using sur- face sterilized AMF spores in combination with a post vitro rooting procedure. Besides pathogens, there is also a risk of insect pests, and therefore it is equally important to control the sanitary sta- tus of AMF inoculum as it is to control the phy- tosanitary status of plant material (Cassells 1991). Referring to the above, the importance of an optimal rooting method and the correct timing of inoculation must be emphasized. The carbohy- drate source of the rooting medium has a great effect on the in vitro rooting of apple microcut- tings (Moncousin et al. 1992). Rooting experi- ments conducted with apple cv. Compact Spartan showed that sucrose was an indispensable com- ponent in the agar gelified rooting medium (R Druart, pers. commun.). Uosukainen (1992) showed that both sucrose and glucose in the root- ing medium, combined with optimal auxin treat- ment, resulted in a high rooting rate with apple rootstock YP. The present study showed that the carbohydrate source of the rooting medium has also a great effect on weaning survival of the microcuttings, when in vitro rooted plantlets are used. The highest weaning and survival rates of inoculated rooted plantlets were obtained by us- ing sucrose in in vitro rooting medium. Macronutrients are important to root induction, root emergence and root growth, but high salt levels, high nitrogen levels in particular, are fre- quently inhibitory to root initiation (George and Sherrington 1988). Rooting experiments with the apple cv. Compact Spartan showed that NH 4 NO was an inhibitor of root emergence and further growth. Decreases in average root number/ plantlet, smaller roots and a lower rooting rate were typical. On the other hand, the macronutri- ents KH 2P04 and, particularly, Ca(NO,) 2 stimu- lated root growth (P. Druart, pers. commun.). However, maximum AMF root colonization and sporulation occur in soil of low fertility. Both phosphorus and nitrogen may significantly reduce root colonization if present at high levels and there exists a balance between these two elements (Daniels Hetrick 1986). Thus, there is a contra- diction between the optimal conditions for root- ing and AMF root colonization. This contradic- tion can most likely be reconciled only by using intermediate conditions rather than optimal con- ditions for one or the other partner of the symbi- osis. For example, AMF inoculation after the weaning stage, instead of immediately after the in vitro stage, may give a more positive growth response, as was found by Azcon-Aguilar (1994) in Annona cherimola. The level of mineral nutri- ents in the weaning substrate also needs to be modified to meet the requirements of the devel- oping root systems and undisturbed growth of apple microcuttings. These elements must be fur- ther examined and their effects must be control- lable before AMF inoculation can be introduced to nursery men. Acknowledgements. We are grateful to the Ministery of Agriculture and Forestry for the financial support of this study. We wish to express our special thanks to Mrs. Anja Manninen, laboratory technician Pirkko Jalkanen and ag- ricultural engineer Leena Eerola, for their skilful techni- cal assistance. References An, Z. Q., Shen, T. & Wang, H.G. 1993. Mycorrhizal fungi in relation to growth and mineral nutrition of apple seedlings. Scientia Horticulturae 54: 275-285. Avanzato, D., Cherubini, S. & Jaque, E. 1993. Portain- jertos de manzano de obtenciön in vitro. Potencialidad de la aclimataciön y enraizamiento directo de estaquil- las. 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Manuscript received February 1994 SELOSTUS Arbuskeli-mykorritsasiirrostuksen vaikutus mikrolisättyjen omenapuiden (Mains ) juurtumiseen, viljelyolosuhteisiin sopeutumiseen ja sopeutumisvaiheen jälkeiseen kasvuun Marjatta Uosukainen ja Mauritz Vestberg Maatalouden tutkimuskeskus Maatalouden tutkimuskeskuksen Laukaan tutkimus- ja va- liotaimiasemalla tutkittiin eri juurrutusmenetelmien sekä arbuskelimykorritsa- eli AMF- siirrostuksen vaikutuksia omenapuun mikropistokkaiden juurtumiseen, kasvihuone- viljelyyn sopeuttamiseen ja sopeuttamisen jälkeiseen kas- vuun. Tutkimuksen tavoitteena oli optimoida sekä juurru- tus- että siirrostusolosuhteet siten, että satunnaisten teki- jöiden häiritsevä vaikutus saataisiin mahdollisimman vä- häiseksi ja toisaalta mykorritsasymbioosista saatava hyö- ty mahdollisimman suureksi. Tutkimus toteutettiin neljänä kokeena. Koekasveina oli- vat omenapuun perusrunko YP sekä suomalaiset koriste- omenapuut ’Hanna’ja ’Marjatta’. Tutkittaessa kasvien sel- viytymistä kasvihuoneviljelyyn sopeuttamisvaiheesta to- dettiin, että in v/tro-juurrutusalustan hiilihydraattilähtee- nä sakkaroosi paransi kasvien selviytymistä verrattaessa glukoosi-ja fruktoosipitoisilla alustoilla juurrutettuihin ver- soihin. Paras juurrutus-ja sopeuttamistulos saatiin kuiten- kin juurruttamalla mikropistokkaat suoraan turvepohjai- sella kasvualustalla in vifro-juurrutuksen sijasta. AMF- siirrostus ei parantanut juurtuvuutta suorajuurrutuksessa. Glomus hoi kanta V9B aiheutti vaikea-asteista mikropis- tokkaiden tyvien mätänemistä ja täten jopa heikensi 'Han- na'-lajikkeen juurtuvuutta. AMF-siirrostus kuitenkin lisä- si juurtuneiden kasvien keskimääräistä verson tuorepai- noa, erityisesti siirrostus kannoilla G. hoi V9B, G. claroi- deum V43a tai G. fistulosum VI2B. Tämä vaikutus vaihte- li huomattavasti eri isäntä-sieniyhdistelmissä, ja mm. AMF- kanta G. hoi VIO4 jopa heikensi YP-perusrungon pikku- taimien kasvua. Omenapuun mikropistokkaille oli tyypil- listä, että taimien kasvu pysähtyi juurrutus- ja sopeutta- misvaiheiden jälkeen. Sekä YP- perusrungon että koriste- omenapuiden AMF-siirrostetuilla taimilla tämä ilmiö jäi selvästi harvinaisemmaksi kuin siirrostamattomilla taimilla. Tasalaatuinen taimiaines, optimaalinen AMF-siirrostuk- sen ajankohta sekä juurrutus-ja sopeuttamisvaiheessa käy- tettyjen kasvualustojen ravinnetaso ovat onnistuneen tu- loksen kannalta kriittiset tekijät. Näiden tekijöiden vaiku- tukset on hallittava ennenkuin AMF-siirrostuksella saa- daan aikaan toimiva symbioosi. 279 Agricultural Science in Finland 3 (1994)