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. their origin, but the size of the spores pro- ment. Mycorrhizal leek plants and the four Leek plantlets previously germinated in duced in vitro was significantly smaller (P # monoxenically cultured petri plates mentioned autoclaved sand trays were transplanted to 0.05). Spores produced in vitro had a mean were used as inoculum sources for the exper- 100-mL individual containers filled with diameter of 83 ± 9 mm, whereas those pro- imental setup. Because preliminary analyses pasteurized (100 C, 2 h, twice) sandy soil duced in vivo had a mean diameter of 117 ± showed that the mycorrhizal colonization in (950 g·kg–1 sand, less than 2.5 g·kg–1 organic 13 mm, and spore size distribution indicated the roots of both the leek plant culture and the matter, pH 8.2, 2.0 mg·kg–1 Olsen-P), and clear variations between both samples. The Ri T-DNA transformed carrot roots in the in the different inoculation treatments were diameter of spores produced in vitro varied vitro cultures was similar and above 80%, we applied under the plant roots at transplant. between 66 mm and 105 mm with the highest sought to apply equivalent amounts of poten- Containers, kept for 10 weeks in a greenhouse frequencies between 70 and 89 mm, whereas tial mycorrhizal propagules originated in vivo (20 to 28 C and 135 mmol·s–1·m–2 16 h·d–1), the in vivo spore diameters ranged between and in vitro, and inoculation treatments were received weekly applications of Hoagland’s 90 mm and 145 mm with the highest frequen- as follows. solution without phosphorus and irrigation cies between 100 and 134 mm. Spores pro- In vivo inoculation treatments consisted was controlled daily. There were six replicates duced in vitro were less pigmented and more of: 1) bulk inoculum: 2 mL of substrate (with for each treatment and we carried out two homogenous in size and color. Concerning 50% pore space) from the host plant rhizo- consecutive experiments within a 4-month the colonization density in roots, in plant root sphere, which contained 50 ± 15 spores/g and period. The percentage of root colonization fragments, there were 88 ± 45 intraradical 155 ± 54 mm of root fragments varying in was estimated (Giovannetti and Mosse, 1980) vesicles/cm, whereas in vitro roots had 35 ± shape and size (Newman, 1966) plus external for both subsequent experiments and they 23 spores/cm. mycelium fragments; 2) roots: 10 10-mm root were shown to be reproducible. When my- After a 10-week growth period in a green- fragments excised from the host plant heavily corrhizal root colonization percentage data house, leek plants inoculated with the in vivo colonized root system; 3) spores: 50 indi- obtained in each replicated experiment were treatments had higher mycorrhizal coloniza- vidual spores recovered by wet sieving (pool analyzed as a factorial design, no significant tion rates in roots than the equivalent in vitro of all spores retained on 250-, 125-, and 50-mm mesh sieves) of the bulk inoculum; and 4) sieving: 20-mL aliquot of a distilled water suspension of fungal material recovered after sieving a 2-mL sample of in vivo bulk in- oculum through a 125-mmmeshsieve. In vitro inoculation treatments consisted of: 1) bulk inoculum: 1-mL portions excised from the MSR medium root compartment, which contained 70 ± 21 spores and 284 ± 83 mm of heavily colonized root fragments varying in shape and size (Newman, 1966) plus external fungal mycelium; 2) roots: 10 densely colonized mycorrhizal 10-mm root fragments excised from the same compart- ment after accurate observation under a stereo- microscope (·80 magnification) to confirm mycorrhizal colonization; 3) spores: 50 in- dividual spores obtained after liquefying the medium in the root-free compartment with sodium citrate buffer as mentioned previously; and 4) sieving: 20-mL aliquot of a distilled water suspension including fungal material recovered after sieving through 125 mm a sus- pension obtained after dissolving the agar from the root-free compartment of a monox- Fig. 1. Distribution of diameter values of two populations of 100 Rhizophagus irregularis spores recovered enically cultured petri plate. from in vivo or in vitro cultures.

898 HORTSCIENCE VOL. 48(7) JULY 2013 inoculation treatments (Fig. 2A). Among the Among in vivo-obtained propagules, mycor- smaller diameters than soilborne spores; the in vivo treatments, bulk inoculum, root frag- rhizal root fragments were the most efficient mean diameter value of mature G. etunicatum ments, and sievings were significantly more at stimulating shoot development followed spores formed in vitro was 96 mm, whereas the efficient than individual spores at colonizing by the bulk inoculum treatment. They both mean diameter of spores recovered from pot the root system, although only bulk inoculum significantly increased the growth of leek cultures measured 106 mm. In the present and root fragments infected all the inoculated plants when compared with the noninocu- work, the differences found in R. irregularis plants (12). When 125-mm sievings were used lated control (Fig. 2B). The application of sporesizeweregreaterwithameanincrease as an in vivo inoculum source, 11 of 12 leek nonmycorrhizal inoculum leachates and of of 34 mm in spores obtained from plant plants were mycorrhizal, whereas only nine of nonmycorrhizal root fragments had no influ- culture, corresponding to a 40% gain. One 12 plants became infected in the inoculation ence on plant growth because no significant possible reason for the smaller size of in vitro- treatment with individual spores. Among in differences could be reported when these produced spores is depletion of nutrients or vitro propagules, bulk inoculum was the sole treatments were compared with the noninocu- carbon in the root organ culture. Previous in vitro inoculum source able to colonize all lated control treatment, showing that all dif- work has shown that replenishing carbon or 12 inoculated plants. Nine plants became ferences in plant growth were the result of the nutrients can increase spore number (Douds, mycorrhizal when inoculated with in vitro mycorrhiza. 2002), but information on spore size was not obtained root fragments or spores, and only provided. Spore size has shown a linear re- seven were infected after inoculation with Discussion lation to its number of nuclei (Marleau et al., 125-mmsievings. 2011), a characteristic that hypothetically Plant growth measured as shoot dry weight The significantly different sizes of R. irreg- could be linked to the potential of the fungus showed a response only to in vivo-produced ularis BEG 72 spores produced in either in vitro to develop after germination and to establish inocula. None of the inoculation treatments or in vivo systems indicate distinct adapta- the association with the plant. We observed based on in vitro-obtained propagules stimu- tions of the fungus to the growth medium. that in vitro spores were, in general, of lighter lated plant growth when compared with the Pawlowska et al. (1999) also observed that color, confirming differences in color intensity noninoculated control (Fig. 2B), and no dif- spores of Glomus etunicatum Becker & resulting from the production system, as re- ferences were detected among them either. Gerdemann from monoxenic cultures exhibited ported by Fortin et al. (2002). All in vivo-produced inoculum treatments achieved higher colonization rates as com- pared with inocula produced under in vitro conditions using Ri T-DNA transformed car- rot roots. Among inocula produced in vitro, bulk inoculum, which included all kinds of propagules: spores, hyphae, and root seg- ments, achieved the highest colonization rate in leek roots after 10 weeks (above 60%). Nevertheless, it was unable to enhance plant growth, like all the other in vitro treatments (Fig. 2B). Bulk inoculum and mycorrhizal root fragments produced in vivo were the only treatments capable of significantly stimulating plant growth. In vivo mycorrhizal inoculation treatments had high infectivity potentials, whereas the equivalent in vitro treatments were less effec- tive at colonizing the plant’s root system. Although all types of propagules of AM fungi (spores, root fragments, and hyphae) are able to initiate AM symbiosis (Mosse, 1988), the inoculation treatments based on the use of spores alone were not reliable because only nine of 12 plants became mycorrhizal, and sievings also lacked infectivity. A leaching effect in the leek test plant sandy soil con- tainers could have caused a loss in the number of spores and mycelial fragments and an in- direct decrease of the infectivity potential when they were used as the sole inoculum source. Vimard et al. (1999) also observed a drastic reduction in plant colonization when in vitro-produced spores were used in a highly porous substrate as compared with the colo- nization by spores in soil or by root fragments either in soil or in the porous substrate. High propagule density reduces the length of the lag phase in the curve of colonization percentage vs. time, and that is related to a quick spread of the fungus in the root system Fig. 2. Mycorrhizal root colonization (A) and growth of ‘‘carentan 2’’ leek plants (B) inoculated with bulk inoculum, root fragments, spores, and inoculum sievings from monoxenic culture (in vitro) or from (Smith and Read, 1997; Vimard et al., 1999). plant culture in association with a host plant (in vivo). Different lower case letters indicate significant The increases in colonization and in plant differences between in vivo and in vitro inoculation treatments (Tukey P # 0.05). Different upper case growth stimulation when root fragments from letters indicate significant differences among in vitro inocula or among in vivo inocula (Tukey P # a pot culture were used as inoculum might 0.05). Data are means of 12 plants (A) and six plants (B). thus be related to the high number of infective

HORTSCIENCE VOL. 48(7) JULY 2013 899 intraradical vesicles of R. irregularis, a mor- and mycorrhizal root fragments were able to and plant pathogens under in vitro conditions. phological characteristic of the species when increase plant growth, one possible hypothesis Mycorrhiza 22:437–447. produced in vivo. Fortin et al. (2002) showed is that benefits to plant growth depend on Bianciotto, V., A. Genre, P. Jargeat, E. Lumini, G. that arbuscules and vesicles are often scarce agents or factors present inside plant roots, Be´card, and P. Bonfante. 2004. Vertical trans- in mycorrhizal Ri T-DNA transformed carrot one of such factors being bacteria associated mission of endobacteria in the arbuscular my- corrhizal fungus Gigaspora margarita through roots despite the production of abundant in- with AMF. Continuous in vitro culture of one generation of vegetative spores. Appl. Environ. tracortical mycelium, suggesting a delay in species of Gigaspora led to the elimination Microbiol. 70:3600–3608. the development of the symbiosis when they of the endobacteria from its large spores Bonfante, P. and I.A. Anca. 2009. Plants, mycorrhi- are used as inoculum. In our experimental (Bianciotto et al., 2004). Such bacteria are zal fungi, and bacteria: A network of interac- conditions, the number of spores observed on important for the fungus development (see tions. Annu. Rev. Microbiol. 63:363–383. in vitro root fragments (35 ± 23 per cm), even review by Bonfante and Anca, 2009), because Butt, T.M., C. Wang, A.A. Shah, and R. Hall. 2006. if seemingly lower than the number of vesicles they affect hyphal elongation and branching Degeneration of entomogenous fungi, p. 213– counted within in vivo roots (88 ± 46 per cm), and have been found in several AMF species 226. In: Eilenbergand, J. and H.M.T. Hokkanen was expected to have an infectivity poten- (Lumini et al., 2007). Rhizophagus irregularis (eds.). An ecological and societal approach to tial similar to that of in vivo root fragments. forms smaller intraradical spores, and the loss biological control. Springer-Verlag, Heidelberg, Germany. However, root colonization achieved by in of bacteria when cultured in vitro might be Camprubi, A. and C. Calvet. 1996. Isolation and vivo root fragments when used as inoculum linked to changes in the symbiotic process. screening of mycorrhizal fungi from citrus was much higher despite the uncertain density The reasons for the phenomena we ob- nurseries and orchards and inoculation stud- of propagules within the plant roots effectively served led to several hypotheses indicating ies. HortScience 31:366–369. used as inocula. Because internal colonization fascinating avenues for fundamental research. Camprubi, A., V. Estaun, A. Nogales, F. Garcia- cannot be accurately assessed without a stain- Our main concern, however, was to control if Figueres, M. Pitet, and C. Calvet. 2008. Re- ing procedure, their mycorrhizal potential is propagules of the same AMF produced in vitro sponse of the grapevine rootstock Richter 110 assumed to be equivalent to the colonization were similar to those obtained from the to inoculation with native and selected arbus- estimated in stained samples from part of the classical plant culture system. cular mycorrhizal fungi and growth perfor- root system. The in vitro systems have been proved mance in a replant vineyard. Mycorrhiza 18: 211–216. The propagules originating from inside in useful for the cultivation and conservation of Declerck, S., D.G. Strullu, and J.A. Fortin. 2005. In vivo root fragments, in this case spores, vesi- a large number of species and isolates of AM vitro culture of mycorrhizas. Springer-Verlag, cles, or hyphae, must have colonized the host fungi (Fortin et al., 2002), being a valuable Berlin, Heidelberg, Germany. root earlier, therefore having stimulated the tool to study fundamental aspects of AM Declerck, S., D.G. Strullu, and C. Plenchette. 1996. host growth by the time plant biomass was symbiosis. They are also a very good system In vitro mass production of arbuscular mycor- measured, which was 10 weeks. On the other for massive production of uncontaminated rhizal fungus Glomus versiforme associated hand, propagules from in vitro inoculants spores and have been adopted both for research with Ri T-DNA transformed carrot roots. Mycol. probably promoted a later colonization of and for commercial purposes. Nevertheless, Res. 100:1237–1242. roots with the resulting lack of response in the diverging behaviors in colonization and in Diop, T.A. 2003. In vitro culture of arbuscular plant growth. This hypothesis is supported by growth effects of in vivo and in vitro inocula mycorrhizal fungi: Advances and future pros- pects. Afr. J. Biotechnol. 2:692–697. the delay in early plant colonization observed must be taken into account when extrapolating Doner, L.W. and G. Be´card. 1991. Solubilization of by Vimard et al. (1999), when in vitro spores research results obtained with monoxenically gellan gels by chelation of cations. Biotechnol. were compared with living mycorrhizal plant cultured AM fungi to plant cultured AM fungi. Tech. 5:25–28. roots as an inoculum source. After 16 weeks, Diop (2003) stated that the effectiveness of Douds, D.D. 2002. Increased spore production by levels of colonization achieved by living roots these propagules in natural conditions should Glomus intraradices in the split-plate monox- were still significantly greater than those be investigated, and our results confirm the enic culture system by repeated harvest, gel achieved by in vitro spores, but plant growth need to still increase our knowledge on dynam- replacement, and resupply of glucose to the response was not evaluated in that work. ics of colonization and of exchanges between mycorrhizal. Mycorrhiza 12:163–167. Loss of infectivity along generations of AM fungi and host plants concerning the Elmes, R.P. and B. Mosse. 1984. Vesicular–arbuscular inocula produced in vitro, both for spores and inocula origin. The data reported here indicate endomycorrhizal inoculum production. II. Ex- mycorrhizal root pieces, might reduce the that the production system of AM fungi itself periments with maize (Zea mays)andotherhosts in nutrient flow culture. Can. J. Bot. 62:1531– infectivity and the effectiveness of the sub- can have implications in experimental results, 1536. cultures (Plenchette et al., 1996). This has thus requiring thorough investigation of mech- Ferguson, J.J. and S.H. Woodhead. 1982. Produc- been abundantly reported for pathogenic fungi anisms involved as well as monitoring of tion of endomycorrhizal inoculum, p. 47–54. (Agrios, 2005; Butt et al., 2006; Krokene and responses in cropping systems. In: Schenck, N.C. (ed.). Methods and principles Solheim, 2001; Skovgaard et al., 2002), which of mycorrhizal research. Amer. Phytopathol. Soc., St. Paul, MN. show lower virulence after several monoxenic Literature Cited in vitro culture cycles and also for ectomycor- Fortin, J.A., G. Be´card, S. Declerck, Y. Dalpe´, M. rhizal fungi (Marx and Daniel, 1976). Because Adholeya, A., P. Tiwari, and R. Singh. 2005. Large- St-Arnaud, P. Coughlan, and Y. Piche´. 2002. we used a first-cycle subculture of Ri T-DNA scale production of arbuscular–mycorrhizal Arbuscular mycorrhiza on root-organ cultures. transformed carrot roots infected with the fungi on organs and inoculation strategies, p. Can. J. Bot. 80:1–20. Gerdemann, J.W. and T.H. Nicolson. 1963. Spores AMF isolate, the lower infectivity of these 315–338. In: Declerck, S., D.G. Strullu, and J.A. Fortin (eds.). In vitro culture of mycor- of mycorrhizal Endogone extracted from soil inoculants could not be the result of an aging rhizas. Springer-Verlag, Berlin, Heidelberg, by wet sieving and decanting. Trans. Br. Mycol. effect of in vitro subcultures, therefore seem- Germany. Soc. 46:235–244. ing to be a trait of the fungus when grown in Agrios, G.N. 2005. Plant pathology. 5th Ed. Aca- Gianinazzi, S., H. Schuepp,€ J.M. Barea, and K. root organ cultures. demic Press, San Diego, CA. Haselwandter. 2002. Mycorrhizal technology Another hypothesis is the effect of bacteria Bago, A. and C. 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