33 Lead Lecture
Advances in Arbuscular Mycorrhizal (AM) Biotechnology K. M. Rodrigues and B. F. Rodrigues* Prof. and Head. Department of Botany, Goa University, Goa 403 206 India *E-mail:
[email protected]
Introduction
2.2: Sources of AM inoculum
A rbuscular m ycorrhizal (A M ) fungi from the phylum G lo m e ro m y c o ta a re u b iq u ito u s soil b o rn e m ic ro b ia l sym bionts form ing m utualistic associations with a m ajority of terrestrial plants. T hey fa cilitate uptake of nutrien ts (mostly im m obile P) through their extra-radical mycelial netw ork and provide o th er benefits to their host plants. T hese b e n e fits c an b e p h y s io lo g ic a l, n u tritio n a l and ecological and therefore exploiting and m anaging AM fungi has im portant consequences for both agricultural and natural ecosystem s. N ow adays, they are increasingly considered in agriculture, horticulture, and forestry program s, as well as for environm ental reclam ation, to increase crop yield and health and to lim it the a p p lic a tio n o f a g ro c h e m ic a ls (Gianinazzi et a!., 2002; Johansson et a i , 2004). H ow ever, the obligate biotrophic nature o f AM fungi has com plicated the developm ent o f co st-efficient large-scale production methods to obtain high-quality AM fungal inoculum . T his is one of the reasons why their com m ercial exploitation is still in its infancy (Ijdo e ta l., 2011 ).
A M fungi are obligate sym bionts, grow ing only in association with a host plant. C urrent production system s therefore rely on soil-based system s (plots or pots), which are not sterile and are often contam inated w ith other A M s p e c ie s , a n d o th e r m ic r o b e s , in c lu d in g p a th o g e n s (Gianninazzi and Bosatka, 2004). Non-soil based approaches include in vitro system s involving the use o f Ri T-DNA transform ed plant root organs (genetically m odified w ith A grobacterium rhiz.ogens) to grow on m edia under sterile conditions. T hese are m uch cleaner, but have a lim ited production capacity (D eclerk et a i , 2005).
The inoculum production system s for AM fungi are classified into viz.. the classical sand/soil or m ore advanced su b s tra te -b a se d p ro d u c tio n sy s te m s and the in v itro cultivation system s, which are based either on excised roots i.e. root organ cultures (R O C ) or on whole autotrophic plants ( Ijdo e ta l., 2011).
2.2.1: Soil based systems or pot cultures Soil from the root zone o f a plant hosting A M can be used as inoculum . Such inoculum is com posed o f dried root fra g m e n ts o r c o lo n iz e d ro o t f ra g m e n ts , A M sp o re s, sporocarps. and fragm ents o f hyphae. Soil m ay not be a reliable inoculum unless one has some idea o f the abundance, diversity, and activity o f the indigenous A M species. Spores can be extracted from the soil and used as in-oculum but such spores tend to have very low viability or m ay be dead or parasitized. In such a case, soil sam ple can be taken to set up a 'trap culture' using a suitable host plant to boost the num ber of viable spore propagules fo r isolation, further m ultiplication and also to produce pure or m onospecific cultures.
Am Fungal Inoculum Production and Multiplication: 2.1: Inoculum Production AM fungal inoculum has been utilized in agriculture, horticulture, landscape restoration, and site rem ediation for almost tw o decad es (H am el, 1996). In the early 1990s, researchers described m ultiple w ays in which AM species nanagem em w o u ld be u sefu l fo r su sta in ab le system s, deluding agro-system s and restoration (B ethlenfalvay and inderm an, 1992; P fieger and L inderm an. 1994). In a longrm study com paring organic and conventional agriculture, laeder et ai. (2002) found that AM w ere stim ulated in ■ganic trea tm e n ts, w h ic h w as c o rre la te d to enh an ced >stem health (faunal diversity, soil stability, and m icrobial -ttvity) and to increased crop efficiency.
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Pure cultures or m onospecific cultures are obtained after a know n isolate o f AM and a suitable host are grow n together in a m e-dium (sterilized soil/sand) o p tim ized for developm ent o f AM association and spore for-m ation. It co n sists o f sp o res, c o lo n iz ed root frag m e n ts, and AM hyphae.
2.2.2: Host plant species T he plant grow n to host A M fungi in the inocu-lum production m edium should be carefully selected. It should grow fast, be adapted to the prevailing grow -ing conditions, be readily colonized by A M . and pro-duce a large quantity o f roots w ithin a relatively short tim e (4 5 -6 0 days). It should be resistant to any pests and diseases com m on in the inocula production environm ent.
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34 Gilmore (1968) recommended strawberry (Fragaria sp.) for open pot culture propagation of AM fungi. The range of plant species used since then are too num erous to list. Some com m on tem perate host plants included Zea m ays (com). A lliu m cep a (o n io n ), and A ra c h is hypogaea (peanut). W idely-used tropical hosts included Stylosanthes spp., P a s p a lu m n o ta tu m (b a h ia g ra s s ) and P u e ra ria p h a se o lo id e s (k udzu) (h ttp ://in v a m .w v u .ed u /m eth o d s/ cultures/host-plant-choices ). The host plant should also be fertilized by periodic additions o f a nutrient solution such as H oagland's solution (especially -P) so as to m anage the chemical composition ot the m edium and to regulate the formation o f AM association. To ensure that m ost o f the spores in the inoculum are mature, it is essential to grow the host plant for 12-14 weeks. The m edium is then allow ed to dry slow ly by reducing the frequency of w atering over a week and then withdraw-ing w 'ater co m p le te ly . T he inoculum can then be fu rth er multiplied.
2.3.1: In vitro systems or root organ cultures Ri-plasm id transform ed root cultures w ere pioneered by M u g n ie r a n d M o s se ( 1 9 8 7 ). A n a tu r a l g e n e tic transform ation of plants by the ubiquitous soil bacterium A g ro b a cteriu m rh izo g en es C onn. (R ik er et al., 1930) produces a condition know n as hairy roots. T his stable tra n s fo rm a tio n (T e p fe r, 1 9 8 9 ) p ro d u c e s Ri T -D N A tran sfo rm e d p lan t tissu e s th at are m o rp h o g e n e tic a lly program m ed to develop as roots. T heir m odified horm onal balance makes them particularly vigorous and allows profuse growth on artificial m edia (Tepfer 1989). D aucus carota L. (carrot) and Convolvulus sepiuru L. (bindw eed) were among the earliest species to be transform ed using A. rhizogenes Conn. (Tepfer andT em pé. 1981). For in vitro culture o f AM fungi using Ri T-DNA roots, the disinfected AM fungal propagules (spores and colonized root fragm ents) are plated on to Modified Struilu Rom and (M SR) media for germination after which the germ inated propagules are associated with a c tiv e ly g ro w in g Ri T -D N A tr a n s f o rm e d ro o ts fo r establishm ent o f AM sym biosis (B écard and Fortin, 1988).
2.2.3: Advantages and disadvantages 2.3.2: Advantages and disadvantages Soil-based system s for cultivation o f AM fungi (pots, bags, or beds) are the m ost widely adopted technique for A M fungal inoculum production. Soil-based production system s are the least artificial and support the production of a large set o f AM fungal species (single or m onospecific cultures). In general, they are considered as a convenient system for large-sealc production that is able to reach inoculum densities set for m ass production o f 8 0 -1 0 0 propagules per cubic centim eter (Feldm ann and Grotkass, 2002). In so il-b ased pro d u ctio n system s, the nutrient supplies to the AM fungus and plant can be m onitored and regulated (Lee and George, 2005 ). M ore controlled culture conditions are an advantage as this can lead to insights on factors to optim ize propagule production (Ijdo era!., 2011 ). A disadvantage of soil-based cultivations systems is that, in m ost cases, they cannot guarantee the absence of unw anted contam inants. Besides, these systems are often space consum ing. The AM fungal propagules are isolated through wet sieving and decanting technique, which can be followed by sucrose density centrifugation. In addition to soil or sand as a substrate, technical adaptations such as the addition of glass beads, river sand, or vermiculite have been developed facilitating harvest o f relatively clean AM fungal spores and colonized roots that can be chopped into pieces. The presence o f a substrate, however, provides an inoculum w hich is not directly suitable for m echanical application, as is the case for soil-free production methods (M oham mad et a!.. 2004).
ISBN : 978-81-923628-2-3
The m ost obvious advantage o f in vitro cultivation system s is the absence o f u ndesirable co n ta m in a n ts or m icroorganism s, which m akes them m ore suitable for largescale production of high-quality AM fungal inoculum . While c ross-contam inations by other AM fungi are evidently excluded (if the starter inoculum is m onospecific), the c o n ta m in a tio n by o th er m ic ro o rg a n ism s (e n d o p h y te s. bacteria) m ay o ccur e ith e r at the e sta b lish m e n t o f the cultivation process or at later stages o f culture. T herefore, it may be useful to control the cultures visually, by standard plate-counting techniques and by m olecular techniques. The cultures may be placed in a growth cham ber requiring minimal space for incubation with no light required. The possibility to follow sporulation d ynam ics d uring cu ltiv atio n also provides a m eans to control the level o f spore production and to determ ine the optim al harvesting tim e. Factors that influence optim al production (e.g., nutrient availability, presence of contam inants) can be m ore easily detected and controlled in (liquid) in vitro cultures. As a disadvantage, the diversity (in term s o f genera) o f AM fungi that have been grow n in vitro is low er than under pot cultivation system s. A nother disadvantage o f in vitro production is the costs associated w ith the production system s, requiring skilled technicians and laboratory equipm ents such as sterile w ork flow s, controlled incubators for RO C , and growth cham bers for plant system s. O ther advantages o f the ROC system s are the low requirem ents in the follow -up o f the cultures. Once successfully initiated, the cultures m ay be m aintained for periods exceeding 6 to 12 m onths without intervention (Ijdo eta!., 2011 ). For harvesting o f the in vitro produced AM fungal propagules, solubilization o f the culture
National Conference on New and Emerging Trends in Bioinfomxaties and Taxonomy ‘NETBT2015'
35 nedium is e arn e d out using citrate buffer. The application ;f sterile produced inoculum can be of great value for in ■itro propagation o f high-value crops and ornam ental plants Kapoor et a i , 2008). In addition, in vitro propagation in ssociation w ith A M fungi could reduce m ortality rates and he transplantation shock o f reintroduced endangered plant pecies. It could also be used to enhance the production o f econdary m etabolites used in the pharm aceutical industry Kapoor et a i , 2008). A lthough in vitro cultivation m ethods are currently still costly, it seem s likely that the criteria for quality control o f A M fungal inoculum will result in the u tiliz a tio n o f te c h n iq u e s th a t a re a b le to re d u c e contam ination risks. C ultivation by in vitro m ethods m ay then becom e an im portant m ethod to m eet future quality standards for com m ercial m ass production (Ijdo et al.. 2 0 1 1).
Conclusion L arge-scale production o f AM fungal bio-inoculum is not possible in the absence o f a suitable host due to their obligate biotrophic nature, and it is not possible to identify AM species in their active live stages (grow ing m ycelium ). As a consequence, quality control is often a problem , and tracing the organism s into the field to strictly relate positive effects to the inoculated AM fungus is nearly im possible. In addition, no clear criteria have been set for the quality control o f c o m m e rc ia l in o cu lu m , but m ost likely, the leg isla tio n d e a lin g w ith the a p p lic a tio n o f b e n e fic ia l m icroorganism s will becom e m ore drastic in the com ing decades ( Ijdo et a l., 2011). The production o f AM fungi on plants under in vitro conditions has been recently proposed (Voets et a i . 2005) and extended to hydroponic system s ( Declerck et a i. 2009, W O/2009/090220). Following the pre inoculation o f a suitable autotrophic host plant in the system of Voets et a i {2009). a culture is transferred in a hydroponic c u ltiv a tio n sy s te m fa v o rin g the p ro d u c tio n o f la rg e quantities o f propagules. O ther in vitro m ethods m ight come up. w hich could involve spore p roduction on callus or sp o ru la tio n in s te rile a lg in a te b e a d s o r fu lly c lo s e d hydroponic plant cultivation suitable for the production o f AM fungi ( up-scaling the system o f Dupré de B oulois et a i 2006). H ow ever, other relatively clean m ethods (e.g.. in aeroponics) also have a strong developm ental potential and could be further developed in the future. B ierm ann and U nderm an (1983) already discussed that techniques such as sonication and gradient flotation as well as enzym atic m ethods could be developed to separate intra-radical spores and vesicles from roots. W ith an A M fungus as the only sndophyte. such intra-radical propagules can serve as a high-quality inoculum . The search for and the utilization o f beneficial m icrobes lor sustainable agro-ecosystem s has created a potential for the exploitation o f AM fungi and as such there is a necessity
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to a c c e le ra te th e ir in c o r p o ra tio n as b io fe r tiliz e r s in a g ric u ltu ra l p ro d u c tio n system s. T h erefo re, c ontinuous d e v elo p m en t o f h ig h -q u a lity and lo w -c o st AM fungal inoculum production m ethods is expected, w hich could lead to establishm ent o f more advanced m ethods for large-scale production o f A M bio-inoculum . R e fere n ce s h t t p : / /i n v a m .w v u .e d u / m e t h o d s / c u l t u r e s /h o s t- p l a n tchoices. Bécard, G. and Fortin. J.A. 1988. E arly events o f vesiculararbuscular m ycorrhiza form ation on Ri T-DNA transform ed roots. N ew P hytol 108:211 -218. Bethlenfalvay, G.J. and Linderm an. R.G. 1992. M ycorrhizae in Sustainable Agriculture. Am Soc Agron Special Publication 54. A m erican Society o f A gronom y. M adison. Bierm ann, B. and L inderm an. R.G. 1983. Use o f v esicu lara rb u scu la r m y co rrh iza l roots, in tra rad ic al vesicles and extraradical vesicles as inoculum . N ew Phytol 95:97-105. D eclerck. S.. U do. M .. Fernandez. K.. Voets. L. and de la Providencia, I. 2009. M ethod and system for in vitro m ass production of arbuscular m ycorrhizal fungi. W O/2009/090220 D eclerck. S.. Strullu, D.G. and Fortin, J.A. (Eds). 2005. In vitro culture ofM ycorrhizas, N ew York, NY: Springer Verlag Berlin Heidelberg, pp. 388. Dupré de Boulois. H., Voets. L.. D elvaux. B„ Jakobsen, I. and D e c le rc k . S. 2006. T ran sp o rt o f ra d io c a e siu m by arbuscular m ycorrhizal ungi to M edicago truncatula under in vitro conditions. Environ M icrobiol 8:1926-1934. Feldm ann, F. and G rotkass. C. 2002. D irected inoculum production— shall we be able to design A M F populations to achieve predictable symbiotic effectiveness'.’ In: Gianinazzi S, Schüepp H. Barea JM . H aselw andter K (Eds) M ycorrhizal tech n o lo g y in a g ric u ltu re : from g en es to b io p ro d u cts. Birkhauser. Basel, pp 261-279. Gianinazzi. S. and Bosatka. M. 2004. Inoculum o f arbuscular m ycorrhizal fungi for production system s: science m eets business. Can J B otany 82:1264-71. G ianinazzi, S., Schüepp. H., Barea. J.M. and H aselw andter, K. 2002. M ycorrhizal technology in agriculture: from genes to bioproducts. Birkhauser, Basel. G ilm ore, A.E. 1968. Phycom ycetous m ycorrhizal organism s collected by open-pot culture m ethods. H ilgardia 3 9 :8 7 105. H am el, C. 1996. Prospects and problem s pertaining to the m anagem ent o f arbuscular m ycorrhizae in agriculture. A gr Ecos Environ 60:197-210.
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36 Ijdo, M., Cranenbrouck, S. and Declerck. S. 2011. Methods for large-scale production o f AM fungi: past, present, and future. M ycorrhiza 21 :1 - 1 6 . Johansson, J.F., Paul, L.R. and Finlay, R.D. 2004. Microbial interactions in the m ycorrhizosphere and their significance for .sustainable agriculture. FEM S M icrobiol Eco! 48:1-13. Kapoor, R., Sharma, D. and Bhatnagar. A.K. 2008. Arbuscular m ycorrhizae in micropropagation systems and their potential applications. Sci Hortic 116:227-239. Lee, Y.J. and George. E. 2005. Development of a nutrient film technique culture system for arbuscular m ycorrhizal plants. H o n Science 40:378-380. Maeder, P., Fleissbach, A., Dubois, D., etal. 2002. Soil fertility and biodiversity in organic farming. Science 296:1694-97. M oham m ad, A., M irta, B, and Khan, A.G. 2004. Effects of sheared-root inoculum of G lom us intraradices on wheat grow n at different phosphorus levels in the field. Agric Ecoxyst Environ 103:245-249. M ugnier. J. and M osse, B. 1987, V esicular-arbuscular m ycorrhizal infection in transform ed root-inducing T-DN'A roots grown axenically. Phytopathology 77:1045-1050.
and plant health. The Am erican Phytopatholological Societ\ St Paul, MN: APS Press. Riker, A.J.. Banfield, W.M., W right, W.H., Keitt, G.W. ani Sagen, H.E. 1930. Studies on infectious hairy root o f nursen apple trees. J Agric Res 41:507-540. Tepfer, D. 1989. Ri T-DNA ïvom A grobacterium rhizogenes A Source o f Genes H aving A pplications in Rhizospheri Biology and Plant D evelopm ent, E cology and Evolution In: Kosuge T, N ester EW (Eds. ). Plant M icrobe interactions McGraw-Hill Publishing. New York. pp. 294-342. Tepfer, D.A. and Tempe, J. 1981. Production d ’agropine par des ra cin es fo rm ee s sous T a c tio n d ' A g ro b a c te riu m rhiz.ogenes, souche A4. C R A cad Sci P aris 292: 153-156. Voets. L.. de la Providencia. I.E.. Fernandez, K., IJdo. M., C ranenbrouck. S. and D eclerck . S. 2009. E xtraradical m ycelium network of arbuscular m ycorrhizal fungi allows fast colonization o f seedlings under in vitro conditions. M y co rrh iza l9:347-356. Voets, L„ Dupré de Boulois, H.. Renard,. L„ Strullu. D .G and Declerck, S. 2005. D evelopm ent o f an autotrophic culture system for the in vitro m ycorrhization o f potato plamlets. FEM S M icrobiol Lett 248: 111.
Pfleger, F.L.and Linderm an. R.G. (Eds.) 1994. Mycorrhizae
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