Development of arbuscular mycorrhizal biotechnologyand industry: current achievements and bottlenecks

Miroslav Vostka & Ale Ltr & Silvio Gianinazzi & Jana Albrechtov

Received: 2 October 2012 /Accepted: 30 November 2012 /Published online: 8 January 2013# Springer Science+Business Media Dordrecht 2013

Abstract Advanced scientific knowledge on arbuscularmycorrhizal symbioses recently enhanced potential for im-plementation of mycorrhizal biotechnology in horticultureand agriculture plant production, landscaping, phytoreme-diation and other segments of the plant market. The advan-ces consist in significant findings regarding:newmolecular detection tools for tracing inoculated fungi inthe field;the coexistence mechanisms of various fungi inthe single root system;new knowledge on in vitro physi-ology of the AM fungi grown in root organ cultures;mechanisms of synergistic interactions with other microbeslike PGPR or saprotrophic fungi; discovery of mycorrhizasupportive compounds such as strigolactones. Scientificknowledge has been followed by technological develop-ments like novel formulations for liquid applications or seedcoating, mycorrhiza stimulating compounds or new appli-cation modes. Still the missing components of biotechnolo-gy are appropriate, cheap, highly reproducible and effectivemethods for inocula purity testing and quality control. Alsothere is a weak traceability of the origin of the mycorrhizalfungi strains used in commercial inocula. Numerous poorquality products can still be found on the markets claimingeffective formation mycorrhiza which have very low capac-ity to do so. These products usually rely in their effects onplant growth not on support of host plants via formation of

effective mycorrhizal symbiosis but on fertilizing com-pounds added to products. There is growing number ofenterprises producing mycorrhiza based inocula recentlynot only in developed world but increasingly in emergingmarkets. Also collaboration between private sector and sci-entific community has an improving trend as the develop-ment of private sector can fuel further research activities.Last but not least there is apparent growing pull of themarket and increasing tendency of reduction of agrochemi-cal inputs and employment of alternative strategies in plant-ing and plant production. These circumstances supportfurther developments of mycorrhizal inocula productionand applications and maturation of the industry.

Keywords Arbuscular mycorrhizal fungi . Sustainableagriculture . Inoculum quality . Inoculum tuning .

Large-scale trials/verification . Mycorrhizal technology

1 Introduction

For the majority of plant species including agricultural andhorticultural crops, uptake of water and mineral nutritions,particularly phosphorus, is mediated by the mycorrhizalfungi. As applies to nitrogen (N), the contribution of my-corrhizal uptake and the costs to the plant are still not fullyclear (Smith and Smith 2011). Mycorrhizal fungi have also alot of non-nutritional effects on plant physiology oftenalleviating plant stress caused by biotic and abiotic factors,acting as biocontrol agent, stabilising soil aggregates, pre-venting erosion and influencing plant fittness and sustain-ability of the whole plant-soil system (Smith and Read2008). Many lines of scientific evidence prove not onlyimproved crop yield and resistance of mycorrhizal plantsto environmental factors but improvement of many food-quality properties, such as increased contents of desirable

M. Vostka (*) : J. AlbrechtovInstitute of Botany, Academy of Sciences,Pruhonice, Czech Republice-mail: vosatka@ibot.cas.cz

A. LtrSymbiom Ltd., Lanskroun, Czech Republic

S. GianinazziInoculumPlus Ltd., Dijon, France

J. AlbrechtovFaculty of Science, Charles University, Prague, Czech Republic

Symbiosis (2012) 58:2937DOI 10.1007/s13199-012-0208-9

antioxidants, vitamins and mineral elements (Gianinazzi etal. 2010; Albrechtova et al. 2012). Mycotrophic agriculturalcrops are usually forming symbiosis with mycorrhizal fungiand arbuscular mycorrhiza (AM) is the most common typeoccurring in more than 80 % of plant families. Sustainablemanagement of agricultural ecosystems includes efficientmanagement of soil microorganisms (Jeffries et al. 2003;Selosse et al. 2004; Bunemann et al. 2006; Barrios 2007;Vosatka and Albrechtova 2009; Gianinazzi et al. 2010).

Responding to the need of fast-growing world popula-tion, in the middle of the previous century the GreenRevolution brought development of dwarf and semi-dwarf genotypes of high-yielding varieties of wheat and riceresponsive to higher-fertilisation inputs (Khush 2001; Lynch2007) while improving the yield by high dosage of pesti-cides with multiple environmental haphazards (Pimentel1996). The focus of new green revolution is to improvethe yield of crops grown in infertile soils (Lynch 2007)and attention has to be focused to below-ground crop traitssuch as root architecture including root branching (De Smetet al. 2012), nutrient uptake and nitrogen fixation (DenHerder et al. 2010) and management of beneficial soilmicroorganisms in general and to arbuscular mycorrhiza(AM) in particular (Gianinazzi et al. 2010). Abundanceand diversity of mycorrhizal fungi populations in soilsdecreases with soil degradation, pollution, agricultural prac-tices (e.g. tillage, crop station mode) and agrochemicalapplications (Plenchette et al. 2005; Bunemann et al. 2006;Vostka and Albrechtov 2008). Comparison of AM fungiabundance and diversity indicates a potentially severe lossof ecosystem function under conventional high-input farm-ing compared to more ecologically-friendly low-input agri-culture (Oehl et al. 2004). Particularly, high phosphateavailability in soils has negative effects on mycorrhiza de-velopment and usually no mycorrhizal effects on yield are,thus, recorded (e.g. Janos 2007; Douds et al. 2012). Degreeof mycotrophy plays an evident role particularly when cul-tivation conditions involve either soil-less or fumigatedsubstrates used often in horticulture where indigenous my-corrhizal fungal populations are either lacking or greatlyreduced. Fields in conventional agriculture can have signif-icantly reduced AM populations. In organic agriculturewhere low-input cropping systems are maintained, the roleof mycorrhiza has increased significance in production ofmycotrophic crops in comparison to conventional agricul-ture where mycorrhiza function and importance has beenmarginalised by high inputs of agrochemicals (Harrier andWatson 2003; Plenchette et al. 2005; Gosling et al. 2006).

Scientific knowledge on mycorrhiza effects on variouscrop plant parameters are recently increasing emphasisingpotential use of mycorrhiza application in production sys-tems (Vostka and Albrechtov 2008, 2009; Gianinazzi etal. 2010). Mycorrhizal fungi are widely recognized as a

natural plant health insurance (Gianinazzi and Gianinazzi-Pearson 1988). During the last decade, mycorrhizal inocu-lations are becoming recognized as a modern feasible bio-technology in plant production and the whole industry on itsown with its specifics has emerged (Vostka et al. 2008a,2008c); there is an increasing number of examples of thepositive impact of mycorrhiza in crop production and inparticular AM. Mycorrhizal technology is currently reach-ing an industrial stage supported by extensive applied re-search and commercial applications emphasizing anecological, sustainable aspects of the use of mycorrhiza(Vostka and Dodd 2002; Vosatka and Albrechtova 2009;Gianinazzi et al. 2010).

2 Current mycorrhiza industry achievements

The production systems of arbuscular mycorrhizal fungihave evolved significantly during the last years from rela-tively trivial technologies of in vivo cultivation on the rootsof plants grown in the field in non sterile open nursery orfarm cultures (e.g. Douds et al. 2012) or pot production (e.g.Feldmann and Schneider 2008; Vostka et al. 2008b). TheAM fungal inoculum production in association with the hostin non axenic conditions is simple and relatively cheapmethod, however, it has a disadvantage of limited applica-tion, and it might be easily contaminated and it is not welladapted for the development of an industrial activity(Gianinazzi and Vosatka 2003). Due to simple set up andeasy control, the most abundant AM fungi productionoccurs in greenhouses with different inert substrates withplants inoculated with pure fungal cultures or their mixes incontainers or pots (Feldmann and Schneider 2008; Vostkaand Albrechtov 2008; Vostka et al. 2008b). However, themost important issue about commercial production in in situsystems remains the microbial quality and purity of AMFstrains contained inocula and presence of other contaminat-ing microbes. The purity amd quality of inoculum can bechecked by new-generation molecular sequencing methods(454 pyrosequencing) recently being used for AMF diversi-ty studies (Opik et al. 2009). More sophisticated productionmethods in vitro have been recently devloped e.g. produc-tion on transformed hair root cultures (ROC - root organcultures) in axenic conditions (Declerck et al. 2005, 2011).Currently, there are about 3 small enterprises producing invitro in Europe, two large ones outside Europe, however, theproportion of both types of production modes is difficult toestimate globally, since the real commercial data are notalways publically available.

During the last decade a number of companies manufac-turing mycorrhiza based inocula has increased significantly.By our estimation, there are over 15 small medium enter-prises just in Europe producing mycorrhizal fungi, however,

30 M. Vostka et al.

it is often difficult to distinguish the primary producers ofinoculum from distributors of re-branded products. Previ-ously each company has established its own local market,mainly in their own territories but recently markets havestarted to overlap and several distributors chains have beenformed. Few companies have been bought out by multina-tional corporations and have started to market mycorrhizaworld wide.

Nevertheless, the majority of the companies are smallscale and have focused primarily on retail hobby markets.The major product lines they have launched are small userspackaging for the garden centres. Partly the companiessuceed to engage also professional horticulture market andproved economic feasibility of inoculation for specific mar-ket segments like tree planting at landscaping, high valuefruit trees and shrubs production. Also an important targetsegments for inoculations are nursery production of forestand ornamental trees (Vostka and Albrechtov 2008) andphytoremediation projects where plants encounter severestress imposed by high pollutants contents such as heavymetals or organic pollutants. In general, inoculation out-come is more apparent and thus economically feasible inlow-fertile and arid soils where nutrients and water resour-ces are scarce. Applications of mycorrhizal inoculations inconvential production systems for crops in extensive agri-culture are still disputable for uncertain effects since inocu-lation can be effective but economically unfeasible.

There is a significant move of some companies to devel-op new emerging markets in developing countries. There islittle information available on commercial implementationof mycorrhizal technology and particular projects but it isclear that there has been a lot of applications in severalcountries of Africa, significant development of markets inIndia (Adholeya 2012) and other countries (Kasuya andCosta 2009).

3 New potentials for mycorrhizal research andapplications

Until now the most emphasis has been given on nutritionaleffects of mycorrhiza for plantsparticularly uptake ofphosphorus (P). It is generally known that AM developsand functions more effectively for crop yield, growth andplant resistence under conditions of low availability ofphosphates in the soil for plant uptake. This is particularlyimportant in these days when sources of rock phosphate aregoing to be exhausted in decades from now on and mineralfertiliser production is going to be hampered severely(Hinsinger et al. 2011). There are also many non-nutritional effects on plants and their functioning in ecosys-tems, which might be important and support ecosystemservices provided by mycorrhizal fungi (Barrios 2007;

Gianinazzi et al. 2010). One of the most important ecosys-tem services are enhancement of soil erosion control by soilbinding capacity via mycorrhizal extraradical mycelium(ERM) (Caravaca et al. 2002; Rillig 2004; Piotrowski etal. 2004), and significant allocation of plant carbon productsof photosynthesis into mycorrhizal structures being a signif-icant carbon sink on global level (Gianinazzi et al. 2010).There are also new findings on AM role in mediation ofallelopathy through easier transport mediated common my-corrhizal networks (Cipollini et al. 2012).

Currently, soil microbe management leading to sustain-able management practices belongs to possible applicationsof mycorrhizal or combined-microbial inoculations withever increasing importance. During time of ongoing climatechange desertification of agricutlrural land increases, how-ever, microbe management still plays a secondary role inconventional agricultural practices. One of the reasons is anongoing lack of legislative policy focusing explicitly on soilecosystems and degradation processes, scarce internationalpolicy framework to guide sustainable soil management(Thomas et al. 2012). According to the authors, the UnitedNations Convention to Combat Desertification (UNCCD)remains the only Rio Convention that is not supported byinvolvement of scientific community and lacks the equiva-lent of an IPCC (Intergovernmental Panel on ClimateChange) or the proposed IPBES (Intergovernmental Sci-encePolicy Platform on Biodiversity and EcosystemServices).

Implementation of mycorrhizal inoculum into practice ofphytoremediation (phytoextraction, phytostabilisation) hassome potential (Miransari 2011a; Bhargava et al. 2012;Meier et al. 2012). There are several evidences that certainselected AM fungi have capacity to reduce translocation ofheavy metals into above-ground plant tissues and thereforeimprove safety and quality of edible parts of the food crop(Rivera-Becerril et al. 2002).

On the other side, some fungi can increase phytoextrac-tion and then help in decontamination of polluted soils.Nevertheless careful selection of inoculants in this aspectshould be performed prior large scale applications becausesome fungi can have an opposite effects on heavy metaltranslocations (Janoukov et al. 2012).

Newly exploited potential of AM became enhancementof food quality in crop plants. There has been recentlyreports on increase of sugar content in plant, increase ofessential elements (Zn, Mg etc.) and mainly antioxidantsand beneficial mineral elements (Perner et al. 2008;Albrechtova et al. 2012; Gianinazzi et al. 2010).

Currently, a lot of attention has been devoted to theresearch on the impact on interactions between mycorrhizalplants and pathogens, herbivores, and parasitic plants (Junget al. 2012). Modulation of plant defense responses as aconsequence of mycorrhiza establishment results in a mild,

Development of arbuscular mycorrhizal biotechnology and industry 31

but effective activation of the plant immune responses notonly local but also systemical (Jung et al. 2012; Hao et al.2012). An interesting issue in mycorrhizal plant-parasiteinteraction is a recently reported potential of mycorrhiza toprovide partly protection and control of Striga (Fernandez-Aparicio et al. 2011).

Recently defined phytohormones strigolactones inducesuicidal germination of Striga seeds, control shoot branch-ing and seems to be part of host recognition signals formycorrhizal fungi (Fernandez-Aparicio et al. 2011; Kohlenet al. 2011). However, this phenomenon should be furtherinvestigated and eventually exploited since a role of stri-golactones in plant signalling and how strigolactones wereco-opted as an allelochemical signal by parasitic plants stillhas not been fully elucidated (Tsuchiya and McCourt2012). Recently other molecules active in signalling anddevelopment of AM symbiosis have been discovered, e.g.sulphated and non-sulphated simple lipochitooligosacchar-ides (LCOs), and they hold promising potential of AMsymbiotic efficacy enhancement in agriculture (Maillet etal. 2011).

Important issue for development is also combinationswith other beneficial microbes. Synergistic inoculationsbring benefits to plant production, so they are already soughtafter. This, for example, applies to dual inoculations con-sisting of AM fungi and rhizobacteria, which promote plant-growth together (Vazquez et al. 2000; Gryndler et al. 2002;Miransari 2011b). Dual inoculations with AM fungi andsaprotrophic fungi decomposing supplied organic plant mat-ter can exhibit a beneficial effect on the crop growth andfood quality, as we recently proved on mycorrhizal onions(Albrechtova et al. 2012). Members of the saprobic genusTrichoderma have emerged as an especially promisinggroup of microbial inoculants when Trichoderma genotypessupporting plant growth were found (Camprubi et al. 1995;Tchameni et al. 2011). Consistency of given interactions isstill to be assured before safe implementation of the microbemixes.

As examples of newly emerged knowledge based onrecent findings, we can emphasize ecosystem services ofmycorrhiza in agriculture (Gianinazzi et al. 2010), crucialrole of AM in phytoremediation (Bhargava et al. 2012;Meier et al. 2012), new findings on AM role in mediationof allelopathy via common mycorrhizal networks (Cipolliniet al. 2012).

4 Problems and bottlenecks

In commercial AM fungi production, the main issue remainsquality assurance. The only way is to produce pure, non-contaminated AM inoculum is currently in production sys-tems on transformed roots in axenic conditions. However,

still up-scaled mass in vivo production has some limitations,for example not all fungal species can be cultivated success-fully in axenic conditions. In non-axenic production systemsthis issue remains a crucial point in the production and hasbeen dealt with since many years ago (Von Alten et al. 2002;Vostka and Dodd 2002; Gianinazzi and Vosatka 2003;Feldmann and Schneider 2008).

During the in situ host-plant mediated inoculum produc-tion, root samples of host plants used for AM fungi inocu-lum production should be microscopically checked for thepresence of potential pathogenic fungi. Additional test basedon trap plants susceptible to soil-borne pathogens can bealso be used, like cress, because it is very susceptible to rootrots. However, none of these methods is actually satisfactoryand there is an urgent need for molecular tests, for detectingfungal pathogens in AM fungi inocula. Mycorrhizal indus-try is taking the necessary measures in order to ensure thatthe inoculum producers will respect the defined criteria ofquality. For example, the Federation of European Mycorrhi-zal Fungi Producers (FEMFiP; www.femfip.com) has beenfounded (Vostka et al. 2003) with the aims to achieve andmaintain the highest standards of inoculum quality. Also,the internationally recognised IMS (International Mycorrhi-zal Society, www.ims.com) is becoming very important,which among others also has an aim to help corporationsproducing inocula in promoting their products name andquality. Nevertheless it still remains responsibility (at leastmoral and selfdefending ones) of scientific community toserve as watchdogs of the mycorrhizal products fairness andquality.

Following mass production, fungal propagules must beformulated in such a way that they can be stored anddistributed under a wide range of temperatures and withoutloosing viability for quaranteed period. Formulation shouldbe simple, economic, and the inocula should be easy totransport and apply. Some companies have adopted theapproach of single formulations for all markets, while othersproduce a range of products for their target buyers. Howev-er, inoculum tuning seems to be necessary in particularwhen targeted to more extreme conditions like arid, salinesoils, polluted soils or climate zones with distinct features(Vostka and Dodd 2002).

The form of final product implies also the consequentmode of application. Dry formulation of inocula can behomogenised into a cultivation substrate or placed as a layerbelow the seeds. It can also be applied as a gel formulationon bare root seedlings or root balls or even cuttings. Thereare several products on the market in a liquid form, howev-er, viability of the AM fungal propagules in non-sterileliquid media is generally very short, usually only few days,since bacterial and mold contamination builds up in fewdays (Vostka M, Ltr A, Kolom P, unpublished resultsof experiments conducted in the scientific division of the

32 M. Vostka et al.

Symbiom company.). For example, commercial micro-propagated plants can be inoculated post-vitro (Vestberg etal. 2002; Gryndler et al. 2002) straight at the transplantationstage or bare roots plants can be dipped into gel formula-tions of mycorrhizal inocula before transplantation, or thedry formulations of inocula can be spread into the plantinghole. For large-scale application, machinery is needed, i.e.mixing tanks for application in substrates or sowingmachines for field applications.

One of the main issues will always be economical feasi-bility of mycorrhizal applications especially in low valueand extensive crops. Cost benefit ratio of different applica-tions is crucial for any large scale use of mycorrhiza. Sav-ings on fertilizers, water, pesticides should be comparedwith increased yield of target crops and cost of inoculumand inoculum application should be taken into account.Cost/benefit calculations for commercial rose production(production facilities size about 100 ha) showed that byreducing NPK to 50 % and application of the AM fungi atplanting time, a total 267 Euro is saved per hectare permonth (cost of AM fungi is calculated 2 Euro per L) andresulting saving is about 1.5 M Euro for commercial pro-duction of roses in Kenya (Vostka M, Maimba F, unpub-lished results on rose production in Kenya.). The inoculationis, however, too costly for extensive crops like cereals.

Main problem of inoculations is that it is not always work-ing and there might be lack of consistent positive results infield applications. Sometimes present native fungi are abun-dant in target cultivation systems and they can be sufficientlyeffective and costs of inoculation is not outbalanced by theoutcomes. Advances in molecular techniques enable to proveeffectiveness of AM fungal inoculation expressed by improvedyield even in high presence of indigenous populations of AMfungi in the field soil (Hernadi et al. 2012; Pellegrino et al.2012; Sykorova et al. 2012). Future research should focusedmore on the interaction of introduced and native mycorrhizalpopulations and on tracing of inoculated strains to determinetheir persistence of inocula in field soils and their effectivenessin forming colonization with either annual or perennial crops.

Another problem often encountered in mycorrhiza ap-plication is that in conventional practice of plant produc-tion there are numerous agricultural practices (e.g.agrochemical applications, tillage, cropping systems),which inhibit mycorrhiza development, particularly in topsoil layers (Oehl et al. 2004, 2005). There are concernsparticularly on the use of pesticides. Insecticides and fun-gicides (particularly systemic, copper ones) have beenreported to exhibit many negative effects on soil organismswhereas few significant effects of herbicides have beendocumented (Bunemann et al. 2006). Another concernarose based on new findings on the role of mycorrhiza inallelopathy via common mycorrhizal networks (Cipolliniet al. 2012). Authors suggest, that implications of

microbial reintroduction (or controlled inoculations) inagricultural systems on allelochemical modification shouldbe explored, since fungal network seem to serve as high-ways for allelochemical movement directly from donor totarget plants. But it is still unclear how important thismechanism can be in field conditions.

Other aspect of different mycorrhizal responsiveness ofcommon crops currently under consideration is orientationof selection and breeding programs during the last centurytowards maximum plant performance in high-input produc-tion agroecosystems and concern of selection of modernlines towards phosphate uptake without AM fungi (Sawerset al. 2008). As an example is often given wheat (Triticumaestivum) varieties developed before and after 1900, moreand less responsive to AM colonization, respectively(Hetrick et al. 1992). However, recent study based on ameta-analysis on 39 studies from 1981 to 2010 (Lehmannet al. 2012) stated that new crop plant genotypes did not losetheir ability to respond to mycorrhiza due to agricultural andbreeding practices.

The development of an industrial activity producing mi-crobial inocula is a complex procedure that involves notonly the achievement of the necessary biotechnologicalknow-how, but also the ability to respond to the specificallyrelated legal, ethical, educational and commercial require-ments (Gianinazzi and Vosatka 2003). There are numeroussnake oils (unreliable products) on the market and not allmycorrhizal products are capable to enhance effective my-corrhizal symbiotic associations (Tarbell and Koske 2007).They are often labelled as mycorrhizal inoculum and listnumerous AM fungi (and other mycorrhizal fungi, bacteriaetc.), however, true content is different and presence ofviable microorganisms is very low or none. In the 10- weektest of 5 different commercial inocula performed repeatedlyon maize it was shown that some inocula used in dosagerecommended was capable to enhance maize growth in inertsubstrate (autoclaved zeolite) fertilized only by Hoaglandsolution once a week (Fig. 1a), however, only two out of 5products were capable to form any mycorrhizal colonization(Fig. 1b). It shows that the effects was rather accountableto content of nutrients, aminoacids, humic acids and sim-ilar components rather than development of beneficialmycorrhizal association. Similar failures in testing com-mercial inocula have been reported previously (Rowe etal. 2007). Obviously the end users of the inocula i.e.farmers or horticulturalists have little chance to estimatemechanisms of the effect eventhough it is visible andtherefore with these inferior quality products that justbuy packs of very expensive organic fertilizers whichcan do the job of making plant greener and larger in ashort term but would never provide formation of effectivemycorrhizal symbiosis desirable for longer term and sus-tainable mycorrhizal effects.

Development of arbuscular mycorrhizal biotechnology and industry 33

5 Solutions and ways forward in mycorrhizalbiotechonology?

Mycorrhizal applications in agriculture are dependent onscientific evidence supplied by fundamental and appliedresearch in collaboration with end usersthis is the maindirection of the way forward: deepening of collaboration ofmycorrhizal scientists, mycorrhizal industrial producers andmycorrhiza end users. Challenge for areas with well-developed conventional agricultural cultivation systemsremains selection of appropriate inocula formulations andadjustment to more-mycorrhiza friendly cultivation practi-ces in order to ensure inoculation efficacy.

Furthermore, crop breeding programs could include as-pect of mycorrhizal responsiveness to help to attain maxi-mum benefits following mycorrhizal symbiosis in cropproduction. Recent advances in molecular mechanisms ofAM symbioses (Smith and Smith 2011) and breedingapproaches are helping to optimize the use of AM fungifor improvement of crop productivity. Recent study basedon meta-analysis indicates that in general, new crop plantgenotypes have not lost their ability to form mycorrhiza andthat plant breeders can include new cultivars in their germ-plasms (Lehmann et al. 2012).

To improve consistency in inocula applications there isessential need to ascertain that inocula will be standardizedand will have always the same quality. The development ofinternationally recognized methods of quality assurance isan important step to be taken on the way to achieve stan-dardization of mycorrhizal products.

Based on scientific evidence, to meet various demands ofsoils and climate it is better to adhere to inoculum tuning and touse native fungal strains in inocula formulations (Vostka andDodd 2002). To reduce costs of products the inocula should beoptimally produced locally especially for overseas regions.There are also important concerns about shifting foreign unau-tochtonous inocula between continents (Schwartz et al. 2006)and local production with native strains of AM fungi can helpto solve this problem. There certainly are ubiquitous AM

fungal species found in variable soil conditions of severalcontinents, however, there seems sometimes be evidencedlevel of the AM fungal species specificity towards pH, plantspecies, pollutants level or degree of soil disturbance andinoculum tuning often offers suitable solution (Vosatka andAlbrechtova 2009), what applies also to ectomycorrhizal fun-gal inoculum (Vostka et al. 2008b). The main question iswhether for perrenial crops the resilience of introduced AMfungal strain is sufficient so the inoculated fungal species canstill be found after few years persisting in the root system. Thishas already been proven possible in the field conditions(Sykorova et al. 2012).

An important issue is also to adjust application modessuitable for a given cultivation system. For example, develop-ment of functioning seed coating is essential for large scaleapplication on extensive crops as cereals. Innovation of prod-ucts leading to use of highly concentrated, high quality andefficacy of biological component can substantially enhance useof mycorrhiza in plant production. More intensive applicationmodes via liquid formulation applicable in irrigation systemcan increase substantially feasibility of mycorrhization in hy-droponic cultivation systems. Also retrofitting of mycorrhizalproducts into soil around mature perrenial plants like trees andshrubs via injection can be supporting tool for further increasein mycorrhiza use. It has been proven by molecular tools thatinjected AM fungal strain into root system of mature plant iscapable to colonise root already colonized by other AM fungalspecies (Vostka M, Ltr A, Kolom P, unpublished results ofexperiments conducted in the scientific division of the Sym-biom company) or that two fungal species can coexist andinteract in single plant root system (Janoukov et al. 2012).

There are several supporting aspects for further develop-ment of mycorrhizal technology and that is certainly grow-ing demands for sustainability and organic products. Alsoconstantly increasing cost of fertilizers and future scarcity ofsome fertilizing components like phosphates support devel-opment of biological alternatives.

One of the most important aspects for the development ofmycorrhizal industry is to increase awareness and education

0,0

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Inoc 1 Inoc 2 Inoc 3 Inoc 4 Inoc 5myc

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izal

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izat

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Fig. 1 Test of five commercial AM fungi based products regularlysold on EU and USA markets (Inoc 1 to Inoc 5, their real namesare not disclosed): a Dry shoot weight (g) and b the root mycor-rhizal colonization (%) evaluated on maize after 10-week-

cultivation in zeolite. Bars above columns represent SD, the differ-ent letters above the bars indicate significant differences betwenmeans (n06) P

leading to organic approach as until recent times there was alittle understanding among farmers and potential users. Indeveloping countries, many farms are not using agrochem-icals but only due to the fact that they are either not availableor are too expensive. Intensive agriculture practices areaccepted without adherence to the scientific principles andecological aspects once economic situation allows it toenhance productivity. For professional growers consistentand convincing data are crucial for their decision makingwhether to use or not to use biological alternatives. There-fore good scientific large-scale trials in real practice andeducation of farmers are paramount. Currently each compa-ny in mycorrhizal industry is and should in fact be not only aproducer and marketer but also to certain extent a researchand educational body collaborating with scientists, becauseof rather low awareness of mycorrhizal products.

6 Conclusions

The main way forward in mycorrhizal applications in agri-culture depends on the scientific knowledge derived fromfundamental and applied research and collaboration of bothmycorrhizal science and mycorrhizal industry. That is driv-ing force for advances in safe and effective applications ofmycorrhizal products in agricultural practices.

Population growth together with climate change, on-going soil degradation and increasing costs of chemicalfertiliser is making the need for the envisaged NewGreen Revolution which comprises better exploitationof existing soil resources including proper soil-microbemanagement (Lynch 2007).

There is growing number of enterprises producing inoc-ula based on mycorrhizal fungi and recently not only indeveloped world but increasingly in emerging markets. Alsocollaboration between private sector and scientific commu-nity has an improving trend as the development of privatesector can fuel further research activities. Last but not leastthere is apparent growing pull of the market and increasingtendency of reduction of agrochemical inputs and employ-ment of alternative strategies in planting and plant produc-tion. These circumstances support further developments ofmycorrhizal inocula production and applications and matu-ration of the industry.

Still the missing components of biotechnology are appro-priate, cheap, highly reproducible and effective methods forinocula purity testing and quality control. Also there is aweak traceability of the origin of the mycorrhizal fungistrains used in commercial inocula. Appropriate and acces-sible methods for quality and products certification areparamount as there are numerous low quality inocula occurron the markets. We should be aware that mycorrhiza inoc-ulation is not a Panacea, which is going to sort out all the

problems of plant production. Nevertheless, mycorrhizalinoculation can be a part of a good practice inecologically-friendly plant cultivation, in particular inspecific conditions of degraded, arid and nutrient-poorsoils or horticultural substrates lacking mycorrhiza. Thecrucial point is, however, that protection and propermanagement of native AMF populations in soils is aprimary tool to exploit positive effects of mycorrhizalsymbiosis phenomena.

In summary the achievements of mycorrhizal industry arevery promising, challenges though remain and there arenumerous bottlenecks to solve. Further research is necessaryto facilitate commercial exploitation of mycorrhizal fungi.In overcoming these drawbacks the collaboration of scienceand industry is essential. Appeal is very clear and consist inworking together to support achievements and overcomebottlenecks of the mycorrhizal industry. Main solution isto join efforts of fundamental and applied research, farmersand industry to provide consisting evidence for benefits ofmycorrhiza in the real conditions of plant production. Onlythen it will be possible that mycorrhiza will be considered asa part of good practice in sustainable horticulture and agri-culture and it will also help to select reliable products fromthe market from an inferior ones and help mycorrhizalindustry (and research) to grow.

Acknowledgments The authors are thankful to the manuscript editorand two anonymous referees for all their suggestions and comments,which helped to improve manuscript. The authors acknowledge fund-ing of R&D from TACR TAO2020544, projects funded by the Minis-try of Education, Youth and Sports of the Czech Republic in the EU-coordinated scheme Eurostars MycoDripSeed E!4366, and the projectof the Ministry of Industry and Trade of the Czech Republic FR-T11/299 and the R&D project 9210AAO003S3427 funded by the ConseilRgional de Bourgogne (France). Two co-authors Ph.D. Ale Ltr andProf. Silvio Gianinazzi are employed by mycorrhizal inoculum pro-ducer and inoculum distributor, respectively. They declare that theircontribution to the manuscript is based on purely scientific knowledgeand, thus, does not constitute any conflict of interest.

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Development of arbuscular mycorrhizal biotechnology and industry 37

Development of arbuscular mycorrhizal biotechnology and industry: current achievements and bottlenecksAbstractIntroductionCurrent mycorrhiza industry achievementsNew potentials for mycorrhizal research and applicationsProblems and bottlenecksSolutions and ways forward in mycorrhizal biotechonology?ConclusionsReferences