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Plant Growth Stimulation and Root Colonization Potential of in Vivo HORTSCIENCE 48(7):897–901. 2013. Fortin et al., 2002; Gianinazzi et al., 2002; Ijdo et al., 2011). Some studies on the intraradical AM de- Plant Growth Stimulation and Root velopment in monoxenic roots suggest char- acteristics of internal colonization with similar Colonization Potential of In Vivo structures to those of in vivo soil-grown plants (Bago and Cano, 2005), but direct compari- versus In Vitro Arbuscular sons of host plant responses to these two types of inoculum are not found in the literature. In the present work, inocula of a defined isolate Mycorrhizal Inocula (BEG 72) of Rhizophagus irregularis simul- Cinta Calvet1, Amelia Camprubi, and Ana Pe´rez-Herna´ndez taneously produced, for equal time, in vivo Institut de Recerca i Tecnologia Agroalimenta`ries (IRTA), Patologia Vegetal, and in vitro, were evaluated in terms of host root colonization potential and plant growth Ctra. de Cabrils Km 2, E-08348 Cabrils, Barcelona, Spain stimulation. Paulo Emilio Lovato Depto. de Engenharia Rural–CCA, Universidade Federal de Santa Catarina, Materials and Methods CP 476, 88040-970 Floriano´polis, Brazil The fungus used, obtained from Citrus aurantium L. (Camprubi and Calvet, 1996) in Additional index words. inoculum production, biofertilizer, spore morphology, Glomus intra- northeastern Spain, was isolate BEG 72 (In- radices, Rhizophagus irregularis, root organ culture ternational Bank of Glomeromycota) of Rhi- Abstract. Inoculum of arbuscular mycorrhizal fungi, with growing use in horticulture, is zophagus irregularis (Blaszk., Wubet, Renker produced mainly in two technically different cultivation systems: in vivo culture in & Buscot) C Walker & A. Shussler€ comb nov symbiosis with living host plants or in vitro culture in which the fungus life cycle develops according to 500 bp LSU sequence analysis in association with transformed roots. To evaluate the effectiveness and the infectivity (Camprubi et al., 2008) and formerly known of a defined isolate obtained by both production methods, a replicated comparative as Glomus intraradices (Kruger€ et al., 2012; evaluation experiment was designed using different propagules of Rhizophagus irregu- Stockinger et al., 2009). It was simultaneously laris produced in vivo on leek plants or in vitro in monoxenic culture on transformed cultivated during 6 months in association with carrot roots. The size of the spores obtained under both cultivation methods was first leek (Allium porrum L., ‘‘carentan 2’’) plants assessed and bulk inoculum, spores, sievings, and mycorrhizal root fragments were used and in monoxenic culture on Ri T-DNA to inoculate leek plantlets. Spores produced in vitro were significantly smaller than those (plasmid of Agrobacterium rhizogenes)trans- produced in vivo. Although all mycorrhizal propagules used as a source of inoculum were formed carrot (Daucus carota L.) roots able to colonize plants, in all cases, leek plants inoculated with propagules obtained in (Mugnier and Mosse, 1987). vivo achieved significantly higher mycorrhizal colonization rates than plants inoculated For in vivo inoculum production, leek with in vitro inocula. Inoculation with in vivo bulk inoculum and in vivo mycorrhizal root plants inoculated with R. irregularis were fragments were the only treatments increasing plant growth. These results indicate that grown in 1-L volume containers filled with the production system of arbuscular mycorrhizal fungi itself can have implications in the Terragreen (Oil-Dri Company, Wisbech, U.K.) stimulation of plant growth and in experimental results. for 6 months in a greenhouse under sodium- vapor lamps (temperature 20 to 28 °Cand 135 mmol·s–1·m–2 16 h·d–1). In vitro production Arbuscular mycorrhizas (AM) are the most confirms that commercial inocula sold on consisted of monoxenic cultures of the same abundant microbial symbiosis for the majority international markets are not always able to fungus established in bicompartmented petri of plant species, improving their nutrition and perform the arbuscular mycorrhizal symbi- plates supporting fungal development on mod- fitness. The presence of this mutualistic asso- osis. There are basically three production ified Strullu-Romand medium (MSR) in the ciation in natural and agroecosystems, and systems for commercially available inocu- root compartment and on MSR medium with- especially the role of the fungal partner at lants, including a variety of fungal isolates, out sucrose or vitamins in the root-free com- increasing crop yields and plant health, has formulations, and components: the substrate- partment (Declerck et al., 1996; St. Arnaud stimulated the search for mass production based production system, the substrate-free et al., 1996). Plates were incubated at 25 °Cfor methods. Arbuscular mycorrhizal fungi (AMF) cultivation system (both under in vivo condi- the same duration, i.e., during 6 months. To are unable to complete their life cycle and tions), and the in vitro cultivation system (Ijdo assure that similar material was used, we as- reproduce apart from the host plants, although etal.,2011).Theinvivosubstrate-based sessed mycorrhizal colonization in both the their spores can germinate under different cultivation of AM in container-grown plants host plant root systems and the monoxenically environmental conditions in the absence of a is the traditional and the most widely used grown roots after 6 months by the grid-line host (Giovannetti, 2001). Inoculum produc- technique for AM fungal inoculum produc- intersect method (Giovannetti and Mosse, tion has been improved in the last decades, tion. It is useful for large-scale production that 1980). but a recent article by Vosa´tka et al. (2012) requires little technical support. The in vivo Aiming to test a possible influence of the substrate-free system is based on hydroponics production method, we assessed spore diam- and aeroponics, but it has been limited to a few eter size, because previous observations on fungal species. The in vitro system allows spore morphology indicated possible differ- Received for publication 8 Apr. 2013. Accepted for obtaining sterile propagules with high poten- ences between spores produced in vitro and in publication 22 May 2013. tial for research and high-quality inoculum vivo. Individual spores were extracted from We thank Dr. Christopher Walker for helpful while lacking other microorganisms. This the soil rhizosphere of a 6-month-old leek advice on the taxonomy of the arbuscular mycor- method has been widely tested and adopted plant culture by wet sieving (250-, 125-, and rhizas fungal isolate used in this work. We acknowl- by mycorrhizal research laboratories around 50-mm mesh) and decanting (Gerdemann edge financial support from the Spanish Ministry of the world (Declerck et al., 2005). Several re- and Nicolson, 1963) and also from the root- Economy and Competitiveness MINECO grant AGL2010-15017. Paulo E. Lovato had a fellowship views describe the methods for large-scale free compartments of four petri plates with from CAPES Foundation, Ministry of Education production of AMF and point out their poten- 6-month-old monoxenic cultures after lique- of Brazil, Brası´lia/DF–Brazil. tials and limitations (Adholeya et al., 2005; fying the medium with sodium citrate buffer 1To whom reprint requests should be addressed; Declerck et al., 2005; Diop, 2003; Elmes and (Doner and Be´card, 1991). One hundred spores e-mail [email protected]. Mosse, 1984; Ferguson and Woodhead, 1982; from each origin were included in an acid HORTSCIENCE VOL. 48(7) JULY 2013 897 glycerol solution (90 mL distilled water, 10 A noninoculated control treatment was differences (P # 0.05) between them could be mL 1% HCl, 100 mL glycerin) individually included in the experimental design. To verify detected. Therefore, 12 replications per in- placed on a glass slide and measured under possible nonmycorrhizal effects of other in- oculation treatment in a single experimental a compound microscope. Two perpendicular oculum components on plant growth, after design were considered for statistical analysis transversal measures were recorded for each harvesting Expt. 2, four additional control in two one-way analyses of variance (ANOVAs) spore and their arithmetic mean value was treatments were compared with the former (P # 0.05), comparing first the origin of the the variable used in statistical analysis. A one- control treatment: 20-mL leachates free of inocula (either in vivo or in vitro) and second way analysis of variance (P # 0.05) indicated mycorrhizal propagules from in vivo and in the type of propagules used as an inoculum no significant differences among the size of vitro bulk inoculum water suspensions, non- source. At the end of the second experiment, spores produced in the four petri plates and mycorrhizal leek roots, and nonmycorrhizal plant shoot dry weight was determined. Re- because differences resulting from the mon- transformed carrot root fragments. sults were statistically analyzed by ANOVA oxenic cultures themselves could be discarded, To estimate the colonization density in and means were compared with Tukey test we compared a single sample of 100 spores roots, the number of propagules in 20 10-mm (P # 0.05). produced in vitro with the sample of spores root fragments from a 6-month-old leek cul- produced in association with a living plant. ture in Terragreen and from a 6-month-old in Results We carried out a comparison experiment vitro culture of R. irregularis (BEG 72) were to evaluate the influence of both inocula pro- counted under the stereomicroscope (·80 mag- Figure 1 shows the spore size distribution duction methods on their effectiveness in root nification). Plant root samples were previously of 100 R. irregularis spores obtained in colonization and growth stimulation of leek cleared and stained (Koske and Gemma, 1989), monoxenic culture in petri plates or in host plants. The experiment was replicated within whereas structures inside in vitro roots, visible plant culture in the greenhouse. Spores had a 4-month interval and plant growth was without any processing, were observed and a similar morphological shape regardless of monitored at the end of the second experi- counted with no clearing nor staining.
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