Mycorrhiza application in sustainable agriculture and natural systems

Transcription

1 Mycorrhiza application in sustainable agriculture and natural systems Working groups 2 and 4 meeting September Aristotle University of Thessaloniki, School of Forestry and Natural Environment, Forest Soil Lab. Thessaloniki, Greece Organising committee: Jacqueline Baar, Victoria Estaun, Ibrachim Ortas, Michail Orfanoudakis, Dimitrios Alifragis Hotel Philippion 102

2 environmental factors on the development of these symbiontic fungi. If the set-up of such studies is systematic, statistical analysis methods can be applied relating biological components with abiotic factors providing data that are useful for the production and application of AM fungi. In such an approach, environmental factors are of major importance. For mycorrhizal studies, there are two major groups of environmental factors that can be distinguished clearly affecting the development of the fungi. One major environmental factor in mycorrhizal symbiosis comprises the host plant, and the other major factor is the soil environment (see fig 1). Environmental factors host plant soil Systematic set-up Abundance and diversity of arbuscular mycorrhizal fungi Fig. 1. Systematic studies on the relations between the environmental factors host plant and soil and AM fungi provide more insight in the development of AM fungi enabling optimization of production and application of AM fungi. The environmental factor host plant An essential environmental factor in mycorrhizal symbiosis is the host plant. In fact, the symbiosis is as much dependant on the host plant as on the fungus. Approaching mycorrhizal symbiosis from the plant, the plant influences considerably the effectiveness of the symbiosis by its susceptibility. In various experiments, it has been shown that plant species vary greatly in their responsiveness to mycorrhizal fungi (Van der Heijden et al. 1998). Also, crops can vary in their mycorrhizal responsiveness as has been reported for a variety of agricultural crops (Ryan & Graham 2002). For agricultural purposes, breeding programs have resulted in varieties or cultivars with a range of genetic differences. Variation in mycorrhizal dependency of Citrus rootstocks to AM fungi has been reported. However, some of the breeding programs have resulted in poor responses of AM fungi to cultivars of major main crops of economic value. Examples are corn (Zea mays), oat (Avena sativa), barley (Hordeum vulgare) and wheat (Triticum aestivum) (Hetrick et al. 1993; Kaeppler et al. 2000; Ryan & Graham 2002). The breeding of agricultural crops has been carried out for raising resistance to fungal and bacterial pathogens. Unfortunately, successful breeding against pathogens was accompanied with suppression of AM fungal colonization and responsiveness (Ryan & Graham 2002). As a consequence, incompatible reactions between host plants and certain AM fungi have been observed. Hetrick et al. (1993) noted that breeding of wheat cultivars has resulted in high dependency on fertilizers and non-responsiveness to AM fungal colonization. As in many biological processes, numerous genes could be involved in the symbiosis between host plants and mycorrhizal fungi. Experiments with different mycorrhiza-defective plant mutants indicate that root colonization of AM fungi is controlled by a large number of genes (Gollotte et al. 2002). Thus far, most researchers have focused on the identification of plant genes controlling essential steps in the symbiosis between host plant and AM fungus (Balestrina & Lanfranco 2006). A number of the mycorrhiza-regulated genes have been identified by this approach, but there are still plants genes involved in the mycorrhization process unknown (Gollotte et al. 2002). An additional applied approach could be setting up breeding programs aiming for the development of crop varieties that are susceptible for AM fungi. Evidence is growing that wild accessions or old crop cultivars show more susceptibility to AM fungi than modern cultivars of these species, indicating that mycorrhizal responsiveness may have been bred out 2

3 of some crops (Kik, oral. comm.). These studies demonstrate that genetic traits determining mycorrhizal responsiveness exist in the plants as well as in the fungi involved in this symbiosis. This indicates the clear needs of combining the knowledge of plant geneticists and breeders with scientists working on AM fungi in order to develop an understanding of the plant genetic basis for mycorrhizal responsiveness. Cooperation between plant geneticists, plant breeders, and mycorrhizal researchers is one of the aims of the European network COST Action 870 enabling the set-up of programs for searching and developing crop varieties susceptible for AM fungi. Determining differences of various crop varieties in their response to AM fungi fits well into such programs. These can form the basis for developing more optimal combinations of mycorrhizal responsiveness of crop varieties to the most beneficial AM fungi. Further studies are needed to determine the genetic traits in the diverse crops that are of economic value worldwide. Thus far, several studies on the responses of different crops varieties to AM fungi have been carried out, like in The Netherlands (Baar & Ozinga, 2007). Also, some studies for plant breeding have started to study the basis for developing crop varieties susceptible for AM fungi in Europe. Still, more studies on different crops are needed. The environmental factor soil Soil is the other important determining environmental factor for the development of mycorrhizal fungi. This environmental factor comprises different soil factors described either by the abiotic chemical and physical components, or by the biotic soil components. The abiotic factors include chemical and physical composition as well as moisture content of the soil. The vast majority of soils in the world contain AM fungi, but the diversity and abundance can vary. There are some studies showing that soil mineral content and structure can affect AM fungal communities (Johnson et al., 1992; Oehl et al. 2005). Johnson et al. (1992) showed that the occurrence of six AM fungal species was influenced by soil type. The study by Oehl (2005) revealed that the AM fungal communities changed with soil depth and that different AM fungal species were observed in different soil layers. Generally, the development of AM fungi and their effects on plant growth are greater in soils with relatively low nutrient content, particularly with low nitrogen and phosphorus levels, than in soils with relatively high nutrient content. As shown in various studies, high levels of nitrogen and phosphorus, often caused by intensive fertilization with chemical fertilizers and live-stock manure, reduces development of root colonization of AM fungi, but the magnitude of the effect is strongly affected by the fungi studied and by other environmental conditions (Baar & Ozinga, 2007). This is illustrated by a study by Egerton-Warburton & Allen (2000) showing that enhanced soil nitrogen concentrations changed the composition of the AM fungal communities in coastal vegetation communities in southern California and that the abundance of AM fungal spores was reduced by nitrogen enrichment. In a study in The Netherlands, it was found that grasslands with Lolium perenne L. intensively fertilized with live-stock manure with high levels of nitrogen and phosphate for several decades, contained less than 1% of AM fungi and were colonized with oomycetous fungi (Baar & Ozinga, 2007). A more recent study in 2007 showed that colonization by AM fungi in a L. perenne grassland was reduced by intensive fertilization with live-stock manure for over twenty years (see Table 1.). % AC % VC Non-fertilized grassland Fertilized grassland

4 Table 1. Reduced percentage of colonization of AM fungi in grass roots expressed as % AC for the amount of arbuscules and % VC for the amount of vesicles by intensive fertilization with live-stock manure. Samples were obtained in 2007 and colonization levels were determined microscopically after staining according to McGonigle (1990). The studies carried out thus far have provided us with knowledge on the development of mycorrhizal fungi in relation to the chemical and physical composition of the soil. However, the number of studies relating AM fungal development with chemical and physical soil composition in a systematic way is limited (Estaún et al. 2002). Fig. 2. Sampling soil for a study to relate the abundance and diversity of AM fungi to the chemical soil conditions in The Netherlands. Setting up more systematic studies for unravelling the effects of soil variables on the AM fungal development is one of the aims of the European network COST Action 870. If the setup of the studies is systematic, statistical analysis methods can be applied to relate the abundance and diversity of AM fungi with the chemical and physical soil composition. Such systematic studies provide data that are useful for the production and application of AM fungi. Within the European network COST Action 870, new studies are set up for relating the chemical soil composition to the development of AM fungi. References Baar J, Ozinga WA, Mycorrhizal fungi key factor for sustainable agriculture and nature (In Dutch). KNNV-uitgeverij, Zeist, The Netherlands. Balestrini R, Lanfranco L, Fungal and plant gene expression in arbuscular mycorrhizal symbiosis. Mycorrhiza 17: Egerton-Warburton, LM, Allen EB, Shifts in arbuscular mycorrhizal communities along an anthropogenic nitrogen deposition gradient. Ecological Applications 10: Estaún V, Camprubi A, Joner EJ, Selecting arbuscular mycorrhizal fungi for field application. In: Gianinazzi S, Schüepp H, Barea JM, Haselwandter K (eds) Mycorrhizal Technology in Agriculture. Birkhäuser Verlag, Basel, pp Gollotte A, Brechenmacher L, Weidmann S, Franken P, Gianinazzi-Pearson V, Plant genes involved in arbuscular mycorrhiza formation and functioning. In: Gianinazzi S, Schüepp H, Barea JM, Haselwandter K (eds) Mycorrhizal Technology in Agriculture. Birkhäuser Verlag, Basel, pp Hetrick BAD, Wilson GWT, Cox TS, Mycorrhizal dependence of modern wheat cultivars and ancestors: a synthesis. Canadian Journal of Botany 71: Johnson NC, Tilman D, Wedin D, Plant and soil controls on mycorrhizal fungal communities. Ecology 73:

5 Kaeppler SM, Parke JL, Mueller SM, Senior L, Struber C, Tracy WF, Variation among maize inbred lines and detection of quantitative trait loci for growth at low phosphorus and responsiveness to arbuscular mycorrhizal fungi. Crop Science 40: McGonigle TP, Miller MH, Evans DG, Fairchild DL, Swan JA, A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytologist 115: Oehl F, Sieverding E, Ineichen K, Ris E-A, Boller T, Wiemken A, Community structure of arbuscular mycorrhizal fungi at different soil depths in extensively and intensively managed agroecosystems. New Phytologist 2005: Ryan MH, Graham JH, Is there a role for arbuscular mycorrhizal fungi in production agriculture? Plant and Soil 244: Van der Heijden MGA, Boller T, Wiemken A, Sanders IS, Different arbuscular mycorrhizal fungal species are potential determinants of plant community structure. Ecology 79:

6 Introduction about mycorrhizal work in Greece and future possibilities. Orfanoudakis M. Forest Soil Lab, School of Forestry and Natural Environment, Aristotle University of Thessaloniki, Greece PO BOX 271 Introduction The soil is probably the most important natural resource. The soil supports the plant growth by providing the necessary nutrient and water. However the terrestrial ecosystems are losing significant amounts of this unique resource. Human activities such as roads building structures and others lead to loss of surface material, destruction of the organic matter with consequences to several other natural resources and to the atmospheric gases. From the other hand Modern agriculture made possible for large land sites to be in crop production system covering the needs in feeding of an expanding world population. However the modern systems become more depended upon fertilisers, agrochemicals and machinery covering the limited availability in nitrogen phosphate and other plant nutrients (Atkinson et al With such practises significant increase to crop production achieved even at place were the natural ability of the soil to crop production was limited. The desire for maximisation became a necessity. The desire for more quantity easily directs to bad fertilisation management systems. With this significant effect to the agricultural economy, due to the increase spends on chemicals and to the natural resources due to the loss of the extra fertilisers occurred. In addition the bad soil management often led to degradation and to the loss of valuable surface soil material. Such mechanisms could increase to loss of phosphate from the soils. Gradually the soil as natural resource becomes less fertile, unable to sustain the human desire for development. From the times of the antiquities people in Greece were worried by the loss of soil mass and the loss of productivity. (Plato Kritias 111b From Alifragis 2008).These soil changes are common to the Mediterranean regions. The loss of the surface soil leads to a loss of significant AMF number of species. Thus are possible due to the loos of material, increase to the soil temperatures, and the lack of plant species diversity. Farming applications Sustainable agriculture is not just a modern idea about farming but rather a necessity in order to maintain the productivity of the poor framing lands. At a global economy environment to maintain of the maximisation at poor sites is like a dream of a summer night. Managing the soil in favour of AMF diversity is the key to sustainable farming systems. The soil history of the Mediterranean regions however, suggests that the natural occurring AMF population is reduced, significantly, due to the loss of surface soil material or from the extensive use of fertilisers and other agrochemicals. In particular at the second case the remaining AMF population will be from those species fitting better to the environment but with those fitting better to the agrochemicals applied. Selective AMF species application could be an important solution to the problem. The selective species could be imported to the farming system either as selective inoculum or as pre inoculated plants. In both forms could improve the survivability of the host and enchase growth particularly in sites of medium fertility (Orfanoudakis et al unpublished). Natural system applications. Mycorrhizal applications are applicable to the natural systems. As it was prolonged before the degradation is a significant problem of the Mediterranean regions The loss of surface soil layers and the exposure of the raw rock material creates harsh soil condition for the green plants to grow. Heavy metals high soil surface temperatures lack of nutrient availability and the absence of enough water are among the problems should be compensate by vegetation. 6

7 P mg/100g aspercentage of the controls The role of AMF upon such environments is well documented. However there are not enough applications in the field. The management of the natural occurring AMF population could give an advantage. Presumably the autochthonous species are important to the vegetation establishment. Additionally AMF could be applied as selective inoculum and in pre-inoculated plant The effects of the mycorrhizosphere created increasing the availability of phosphate and other nutrients (Fig 1) * * * * * * * Figure 1: Effects extractable P after inoculation with different arbuscular mycorrhizal fungi at the three years of the experiment. Glomus intraradices (empty), Gigaspora margarita (lined), Acaulospora longula (squered), mix of BEG isolates (sphere), indigenous AMF (filled).bars are standard error. Data points marked with an asterisk are significantly different from each other (P < 0.05). Such differences could lead the formation of the plant community. In particular as it is described in Fig 1 the variations of the mycorrhizosphere ability the increase the extractable P from the inorganic soil material could give an advantage to the plants with the highest biological compatibility. Such examples were demonstrated in the past (Van der Heijden et al 1998). Managing this we could drive the natural population to desirable plant diversity. Introduction of exotic AMF species at new sites will eventually turn in favour of the autochthonous fungi. (Qing Yao et al 2007) The lack of enough data upon such mechanism is not well documented and lots of future work is needed on how soon the indigenous species will dominate again or for how long the superiority of selective AMF will occur. Also when applications of selective AMF inoculum occur we need to investigate how interacts with the local plant species. Particularly at natural systems, that could means the AMF application could be in favour of not desirable plant species. References Atkinson, D. and Watson C.A The environmental impact of intensive systems of animal production in the low lands. Animal Science 65: Alifragis D The soil : Genesis-Properties- Clasification. Book I Thessloniki

8 Qing Yao, Hong-Hui Zhu, You-Li Ho and Liang-Qiu Li 2008 Differential influence of native and introduced arbuscular mycorrhizal fungi on growth of dominant and subordinate plants. Plant Ecology 196,

9 What determines a quality mycorrhizal inoculant? Peter Moutoglis BioΣyneterra Solutions Inc., Lanaudiere Industrial and Experimental Carrefour 801 route 344, P.O. Box 3158, L Assomption, Quebec, J5W 4M9, CANADA What determines a quality inoculant? Several presentations have addressed this question over the past years (Adholeya, 2006; Baar et al., 2008; Blal & Parat, 2003; Gagné & Moutoglis, 2006; Gollotte et al., 2008; Gianinazzi & Vosátka, 2004; Moutoglis et al, 2003; Quinn & Blal, 2003) and common ideas that are consistent in all are the following: 1) pathogen-free, non-contaminated inoculant ; 2) inoculant that contains mycorrhizal propagules in the form of spores, colonized roots, hyphae or combinations thereof; 3) methodology(ies) by which the active ingredient (propagules) can be quantified; 4) clearly defined application rates based on statistically significant responses to tested claims; 5) product that has a validated and defined shelf life and storage conditions throughout the supply chain. Most of these criteria are consistent with the Canadian regulatory system which ensures that products are safe for humans, plants and the environment, efficacious for their intended purpose(s) and properly labeled. Efficacy is defined as the ability to fulfill any label claims, supported by scientifically valid efficacy data, and to produce a desired or intended result based on the labeled guarantees and directions for use. This definition includes the ability to clearly demonstrate a benefit to the end user from the application of the product. In addition to specific claims and guarantee(s), each usage pattern or direction for application on the product label should be supported by scientifically valid efficacy data. Anecdotal or testimonial evidence is not to be considered as a scientifically valid form of efficacy data (Government of Canada, Canadian Food Inspection Agency, 2008) but as a highly valuable marketing tool. An example will be presented from field studies of different species of saplings inoculated with ectomycorrhizal inoculants from the selection, production, application, validation and registration process. Small scale nutrient uptake, biomass and stress resistance variables were tested and measured for screening. In addition, the collar diameter and height of the saplings and, in some cases, the dry mass were measured both in small and large scale field experiments. The studies and analyses show that the mycorrhizal inoculant conformed to the required quality standards and resulted in statistically significant responses for the treated saplings. The efficacy data was submitted and the mycorrhizal product was registered for sale in Canada. The ultimate goal was to translate this data into estimates for the potential reductions in greenhouse gases that may result from the mycorrhizal technology which could be registered as Emission Reduction Credits (ERC) and sold to potential clients under a defined carbon trading system. ERC determination was achieved in a three step process. Firstly, the acceleration in growth that results from mycorrhizal inoculation was estimated. Then, the effects the acceleration has on stands were modeled. Finally, the effects from stands to forests were extrapolated. Mycorrhizal technology for inoculating seedlings has been shown, from controlled experiments, to accelerate growth of trees by between 0 and 28%. On average, the acceleration is 10% for Jack Pine, 8% for Black Spruce and 16% for White Spruce (Table 1). This results in substantial increases in sequestration over a rotation. In the simplest situation, at the inoculated stand, we have shown through modeling that inoculation reduces net greenhouse gas emissions by 19.3, 21.2 and 27.4 t CO2e / ha for Jack Pine, Black Spruce and 9

10 White Spruce respectively (Table 2). Extrapolating to the forest, the inoculated stands produce more fiber and decreases harvesting elsewhere in the forest. Modeling suggests that net greenhouse gas emissions would be reduced by 23.5, 24.7, and 31.7 t CO2e / ha for Jack Pine, Black Spruce and White Spruce respectively (Table 3). Mycorrhizal manufacturers, blenders, distributors, retailers and end users have increased over the last five to ten years. The industry is growing at a significant rate in several countries around the world, the majority of which are not regulated, nor do they enforce any quality standards for these types of products. The above case study is but one of many examples that there are good quality inoculants that do what they claim and how they can be applied in innovative ways. Industry can be proactive and along with collaborative efforts with researchers and regulators, a voluntary self-regulating quality system can be conceived and put into effect. This would give rise to increased credibility to the technology and the various products and companies that would voluntarily take part in such an endeavor which would lead to increased acceptance, less skepticism, further research funding, job creation and greater revenues. Such a project could be mediated by an objective, collective, international organization like the International Mycorrhizal Society (IMS) with support from the Mycorrhizal Commercial Relations Committee (MCRC) and COST 870, WG2, Quality control of AM fungal inoculum. Acknowledgement This presentation would not have been possible if not for the close collaborative partnership shared with Mark Kean, Mikro-Tek Inc., Woodrising Consulting Inc. and Canada s climate change Technology Early Action Measures (TEAM). References Adholeya, A In vitro mass production technology for arbuscular mycorrhizal fungi: Scientific and industrial aspects. 5 th International Conference on Mycorrhiza. Mycorrhiza for Science and Society. Granada, Spain, July 23-27, p. 227 Baar, J., Steffen, F., Huig Bergsma, H. & Carpay, B Novel approaches to enhance application of arbuscular mycorrhizal fungi for the development of sustainable agricultural 10

11 and landscape systems: experiences from The Netherlands. Proceedings of COST 870 meeting: From production to application of arbuscular mycorrhizal fungi in agricultural systems: a multidisciplinary approach. Denmark, May 27-30, 2008, p. 27. Blal, B. & Parat J Production, application and regulation of commercial AMF inoculants in agriculture. The Fourth International Conference on Mycorrhizae (ICOM4). Montreal, August 10-15, 2003, p Gagné, S. & Moutoglis, P Challenges for development of mycorrhizal inoculants adapted for specific markets. 5 th International Conference on Mycorrhiza. Mycorrhiza for Science and Society. Granada, Spain, July 23-27, p. 228 Gianinazzi S. & Vosatka M Inoculum of arbuscular mycorrhizal fungi for production systems: science meets business. Can J Bot 82: Gollotte, A., Mercy L., Secco, B., Laurent, J., Prost, M., Gianinazzi S. & Lemoine, M-C Raspberry biotisation for quality plant production. Proceedings of COST 870 meeting: From production to application of arbuscular mycorrhizal fungi in agricultural systems: a multidisciplinary approach. Denmark, May 27-30, 2008, p.11. Government of Canada, Canadian Food Inspection Agency, Trade Memoranda T-4-108: Efficacy data requirements for fertilizers and supplements regulated under the Fertilizers Act. Moutoglis, P., Béland, M., Gagné, S Challenges in commercializing AM inocula in the retail market. The Fourth International Conference on Mycorrhizae (ICOM4). Montreal, Canada. August 10-15, 2003, p Quinn, J. & Blal, B Commercial challenges of ectomycorrhizal fungi. The Fourth International Conference on Mycorrhizae (ICOM4). Montreal, August 10-15, 2003, p

12 Mycorrhizal inoculation of grapevines in replant soils: improved field application and plant performance. Nogales A., Camprubí A., Estaún V., Calvet C. IRTA, Recerca i Tecnologia Agroalimentàries, Ctra. de Cabrils Km 2, E Cabrils, Barcelona, Spain. Introduction Soilborne plant pathogens and abiotic stress factors, such as bad drainage, toxic metabolites or extreme ph are causal agents that contribute to the severity of the vineyard s replant disease. Several species of fungi are associated with the syndrome and among them, the root rot fungus Armillaria mellea (Vahl ex Fr.) Kummer is considered the principal cause of soil fatigue in spanish vineyards. Considering the fact that there are no commercial rootstocks conferring protection in replant situations, few control measures are available. Soil fumigation is banned due to high cost and environmental concerns and long term fallow is strongly recommended before planting, but growers are not willing to wait in intensive production areas. The mycorrhizal inoculation of grapevines under controlled conditions has been achieved by many authors and the beneficial effects on plant growth promotion proved (Linderman and Davis, 2001; Aguín et al, 2004), thus, the use of arbuscular mycorrhizal fungi (AMF) to obtain plants with increased capacity to withstand replant stress has been proposed as a biotechnological alternative. Two consecutive applied research projects, starting in 2000, have been conducted at IRTA, Barcelona, involving growers of several wine production areas in Northeastern Spain. The final purpose was to evaluate mycorrhizal inoculation in replant vineyards by using several inoculation methods, by comparing different AMF isolates, by testing the agronomic response of commercial vine rootstocks of different genetic origin, and by establishing the field performance of mycorrhizal grapevines in replant vineyards with identified replant contributing factors or pathological causal agents. Some of the results obtained are summarized in this presentation, focused on rootstock screening and field growth performance of inoculated mycorrhizal vines. Mycorrhizal inoculation of grapevine rootstocks suitable for mediterranean soils: evaluation of their growth response Five commercial rootstocks tolerant to high lime soil contents and commonly used in mediterranean production areas were inoculated with three Glomus intraradices isolates, two of them obtained from vineyard soils and the registered BEG 72 isolated from similar edaphic and climatic conditions. Hardwood cuttings from Richter 110 (Vitis berlandieri Planch. x Vitis rupestris Scheele), SO4 (V. berlandieri x Vitis riparia Mich.), 41B (V. berlandieri x Vitis vinifera L.), 140 Ruggeri (V. berlandieri x V. rupestris), and 1103 Paulsen ( V. berlandieri x V. rupestris), were rooted in perlite beds (Figure 1) and 15 plants per treatment were either individually inoculated with the mycorrhizal fungi or fertilized with P (0,035 g KH2PO4/Kg substrate) once transplanted to 2 L volume containers filled with a pasteurized substrate mixture (sandy soil, quartz sand and sphagnum peat; 3:2:1, v/v). After six months growth under greenhouse and shadowhouse conditions, plants were harvested and growth parameters measured, and the mycorrhizal colonization achieved was estimated in their root systems. 12

13 Figure 1 Figure 2 Results obtained for shoot dry weight (Figure 3) after the rootstocks screening defined the high mycorrhizal aptitude of the most commonly used vine rootstocks in commercial mediterranean vineyards and the effectivity of the mycorrhizal fungi used as inoculum source. Figure 3 Field experiments Nursery Inoculation of Merlot plants with Glomus intraradices BEG 72 and post transplant growth response in a high lime content replant soil (Calvet et al., 2007) Plants from the cultivar Merlot grafted on the rootstock SO4 were grown in forest pots filled with a sphagnum peat-perlite mixture (1:1,v/v) and inoculated with G. intraradices BEG 72 (Figure 4). Two months later, when 15 plants per treatment were transplanted at random to the field, the mycorrhizal inoculation had caused a significant growth depression in plant shoots, but only five months after the plants establishment in the high lime content replant soil, mycorrhizal plants (Figure 5) outgrew the noninoculated control plants (Figure 6) and their biomass was significantly higher, despite the container s volume used in the nursery. 13

14 Figure 4 One year later, the difference in shoot biomass was still significant between treatments, and moreover, the foliar relative chlorophyll content recorded demonstrated the presence of a higher pigment concentration in plants previously inoculated with G. intraradices. Figure 5 Figure 6 Field inoculation of grapevines in a replanted vineyard soil infested by A. mellea (Camprubí et al., 2008) Cabernet Sauvignon plants grafted on Richter 110 were planted in a high ph replant soil heavily infested by the root-rot fungus A. mellea and with an estimated number of mycorrhizal propagules of 114 in 100 ml. Seventy-five grapevines per treatment were established in the field empty loci left by dead plants previously removed. Four treatments were considered: non inoculated plants, and inoculation with one of the isolates tested in rootstock evaluation (Figure 3), G. intraradices BEG 72 and two native G. intraradices refered as isolate 1 and isolate 2. One hundred grams of fungal inocula developed on Terragreen were placed under the plants, but the traditional planting method involving water flooding around the plants was modified in order to avoid the inocula dispersion and plants were only watered after planting. After 8 months growth, vines were pruned and their shoot biomass recorded. Despite the presence of mycorrhizal propagules in the field soil, G. intraradices BEG 72 significantly increased the growth of plants (Figure 7), while the other two introduced AM fungi did not. The results demonstrated that in the field not all the AMF are equally efficient at increasing plant growth, even if they belong to the same species, and despite their identical performance when they colonized the same rootstock, Richter 110, under controlled conditions (Figure 3). 14

15 Dry weight (g) Figure 7 6 b 5 ab 4 3 a a Control Isolate 1 Isolate 2 BEG 72 Development of new inoculum formulations The implementation of mycorrhizae into the vineyard agronomical practices pointed out the need to adapt the inoculation method to the traditional mechanized planting system. Experimental research has been undertaken to obtain solid formulated products based on the use of biodegradable organic polymers including mycorrhizal propagules which can be easily delivered in the water hole when planting grapevines (Figure 8). Figure 8 Acknowledgements Financial support from INIA ( Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria ) grant RTA C2 and Miguel Torres S.A. is acknowledged. References Aguín O, Mansilla P, Vilariño A, Sainz MJ, Effects of mycorrhizal inoculation on root morphology and nursery production of three grapevine rootstocks. Am J EnolVitic 55: Calvet C, Camprubí A, Estaún V, Luque J, De Herralde F, Biel C, Savé R, Garcia-Figueres F, Aplicación de la simbiosis micorriza arbuscular al cultivo de la vid. Viticultura Enologia Profesional 110: 1-7 Camprubí A, Estaún V, Nogales A, Garcia-Figueres F, Pitet M, Calvet C, Response of the grapevine rootstock Richter 110 to inoculation with native and selected arbuscular mycorrhizal fungi and growth performance in a replant vineyard. Mycorrhiza 2008,18: Linderman RG, Davis EA, 2001.Comparative response of selected grapevine rootstocks and cultivars to inoculation with different mycorrhizal fungi. Am J EnolVitic 52:

16 Use of mycorrhizal inoculum in heavy metal rich industrial wastes Turnau K. 1, Wojtczak G. 1, Ostachowicz B. 2, Ryszka P. 1 1 Institute of Environmental Sciences, Jagiellonian University, ul. Gronostajowa 7, Kraków, Poland, 2 AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Department of Nuclear Methods. Mickiewicza Krakow, Poland Abstract Results of the experiments concerning the use of mycorrhizal inoculum in phytoremediation of heavy metal rich industrial wastes are reported. They include simple AMF inoculation and seeding the area with commercial grass cultivars and introduction of pre-adapted, mycorrhizal seedlings of plants originating from xerothermic grasslands. Heavy metal uptake studies (TXRF) were used to select plant species that exclude most potentially toxic metals from the shoots. Introduction Post-flotation wastes rich in heavy metals are an example of harsh substratum difficult for any biological reclamation (Turnau et al. 2006, Strzyszcz 2003). The slopes of the heap are not only toxic for plants, but also slide down and are extremely difficult to stabilize. The dust originating from the waste heap area often contains high levels of Zn, Pb and Cd which poses serious health hazards for plants and animals. Phytostabilization of such areas is of utmost importance. Typical remediation practices consist of covering the waste with a layer of soil, transported mostly from another area, to prevent erosion. The next step consists of the introduction of trees, and grasses such as Lolium perenne. Although most of these plants are known to form mycorrhiza or other mutualistic associations, typically no inoculation is being carried out. After a few years, especially if the area is not being additionally watered, the soil cover is destroyed and the vegetation is lost, leaving bare spaces of industrial waste. Research on the plants mycorrhizal status occurring on the Zn-Pb waste in Chrzanów has been carried out for the last 15 years (reviewed by Turnau et al. 2006). Several experiments were performed including the introduction of mycorrhizal inoculum followed by seeding various grasses, and also the introduction of mycorrhizal plants originating from xerothermic grasslands. These plants together with the inoculum were introduced directly to the waste without using a soil cover, which is a very expensive practice and demands continuous care and the use of large volumes of water. The main aim was to select proper cultivars or plant species that are able to survive under these harsh conditions (see also Turnau et al 2008) and will avoid the accumulation of toxic elements in shoots. As the nonmycorrhizal plants were mostly not able to survive under such conditions (field and laboratory data) the experiments were carried out focusing on mycorrhiza-assisted plants. Methods Experimental plots were established in autumn 2003 and 2004 in Chrzanów (wastes of the ZG Trzebionka Mining Company). They were devoid of vegetation due to either accidental spills of the sedimental pulp or to mechanical destruction of the surface layer. The tailing material is characterized by 75% carbonate, high concentrations of Ca 2+, SO 4 2- and low Na +, K +, N, P, Mg 2+, Cl - and HCO 3 -. The substratum of the wastes is alkaline (ph 7.4) (Orlowska et al. 2005). Two experiments were carried out. The first one, started in 2003, was carried out on 14 plots each of 20 m 2. A part of these plots was additionally treated with AgroHydroGel (Agroidea Polska). The first seven plots were inoculated each with 15 l of inoculum 16

17 SYMBIVIT (obtained from SYMbio-M, Czech Republic) containing minimum 20 spores per ml of five AM fungal strains (Glomus mosseae CM1, and K1, G. intraradices PH5, G. claroideum BEG 96 and G. etunicatum BEG 136), originating from polluted areas. After inoculation, the plots were covered with an about 5 cm deep layer of substratum and each plot was seeded with one of the following grass species: Lolium perenne L. cv. Inca and Solen, Festuca rubra L. cv. Leo and cv Nimba, Poa pratensis L. var. Alicja, Festuca ovina L. cv. Spartan. The second experiment was started in 2004 and concerned plants originating from dry calcareous grassland located in Kalina-Lisiniec (Wyżyna Miechowska, Southern Poland), members of the following species: Achillea millefolium L., Agropyron intermedium (Host) P. Beauv., Agrostis capillaris L., Anthyllis vulneraria L., Astragalus cicer L., Brachypodium pinnatum (L.) P. Beauv., Bromus inermis Leyss., Cirsium pannonicum (L.) Link, Convallaria majalis L., Dianthus carthusianorum L., Fragaria vesca L., Fragaria viridis Duchesne Inula ensifolia L., Libanotis montana Crantz, Onobrychis viciifolia Scop., Ononis arvensis L., Plantago media L., Verbascum thapsus L. and Veronica spicata L. Two months-old plants that were introduced on the wastes were pre-adapted by cultivation in soil mixed 4:1 v/v with the industrial waste substratum and were planted in rows 15 cm apart. The plants were growing there for almost three years. In October 2007 the shoots (N=5) were collected. Roots were left to allow further growth of the plants. The analysis of metal content in plant material was done using Total Reflection X-ray fluorescence (TXRF) (Hołyńska et al. 1998). For the acid digestion the plant samples were incubated at 185 C for 8 hours. 2 μl of sample were applied onto the clean quartz reflector and measured with a TXRF spectrometer equipped with a Mo X-ray tube. Quantitative analysis was performed using an internal standard (Se). Plate 1. Fig Experimental plots on Zn-Pb Trzebionka waste (Southern Poland). Fig. 1. Grasses growing after introduction of inoculum just before the beginning of dry period. Fig. 2. Verbascum thapsus (in the front row - arrowhead) and Brachypodium pinnatum introduced into experimental plots. Fig. 3. Hieracium pilosella developing within the grass tufts (Festuca ovina). Fig. 4. Verbascum thapsus growing from seeds produced by introduced plants and selfsown within the tufts of Brachypodium pinnatum. Results The introduction of seeds of seven grass cultivars into the metal-rich experimental plots, where the inoculum was applied, was totally unsuccesful. Although the seeds germinated (fig. 1), the seedlings died during the first longer dry period when the plants were ca. 1 month old. 17

18 None of the plant cultivars introduced survived the summer period. Slightly more tolerant to waste conditions were plants on plots treated with AgroHydroGel, but they also did not survive till the autumn. Only a few plants appeared in the plots inoculated with AMF but they were probably from seeds that got to the waste from the surroundings or originated from seeds mixed accidentally into the seed batch used. All these plants were strongly mycorrhizal, although they were not expanding much. In the places where they appeared in the following seasons, some other accompanying plants established, such as Hieracium pilosella and Hieracium aurantiacum. Especially successful was H. pilosella that usually formed new seedlings on the top of the partly dried tufts and the ramets were formed outside (fig. 3). Such ramets sometimes disappeared while it was hot and dry, but after rain they were usually rebuilt from the remaining parts. Also the introduction of xerothermic plants as seeds was mostly unsuccessful, even when mature specimens of grasses transferred from industrial wastes were introduced into the plots to maintain a source of inoculum and to increase the moisture of the place ( nurse plants, fig. 3, 4). Although germination was observed, the seedlings did not survive the large changes of temperature and moisture during late spring and summer. Tufts of grasses transferred from older parts of the wastes were left on the plots, and if they did not perform well themselves, they allowed for the appearance of seedlings of other plant species in the place of the dying tufts. This was observed again in the case of H. pilosella that developed flowers and seeds on the top or close to tufts of Festuca ovina. Both plant species were found to be strongly mycorrhizal and the parameters of the plant performance were similar to populations developing on non-polluted soils. Plants introduced into industrial wastes in the form of seedlings that were pre-adapted by including a fraction of the waste substratum within the soil mix survived better than those cultivated only on non-polluted soil (fig. 2). This was the reason that the pre-adaptation was selected. Most of the introduced plants survived till the end of the experiment. Except for Verbascum thapsus, all other plants propagated vegetatively. They were producing flowers and seeds but new seedlings were not observed during the study period. V. thapsus, a bi-annul plant, was the only one among the introduced species, that spread due to seeds produced by plants introduced into the wastes and formed new seedlings (fig. 4). In laboratory conditions, over 80% of seeds of this plant produced on the waste germinated on wet filter paper within a few days. Among plants introduced into the plots several were efficient accumulators of Pb and Zn. The highest content of Pb was found in the case of V. thapsus (over 1100 mg kg -1 ), Veronica spicata (400 mg kg -1 ), Cirsium pannonicum (360 mg kg -1 ) and Plantago media (300 mg kg -1 ). The highest Zn content was found in V. thapsus (5400 mg kg -1 ), V. spicata (1450 mg kg -1 ), Inula ensifolia (960 mg kg -1 ), C. pannonicum (1700 mg kg -1 ), P. media (1450 mg kg -1 ), Dianthus carthusianorum (870 mg kg -1 ), Fragaria viridis (936 mg kg -1 ). All studied plants contained Zn mostly much above 200 mg kg -1, Pb above 30 mg kg -1 and As (up to 50 mg kg - 1 ). 60% of plant species accumulated also above 70 mg kg -1 of Mn. A relatively low content of potentially toxic metals was found in the case of grasses such as Melica transsilvanica, Bromus innermis, Agropyron intermedium and Anthyllis vulneraria (a member of Fabaceae). Discussion The results presented above showed that simple introduction of mycorrhizal inoculum was not enough in the case of metal-rich industrial wastes. Also the addition of hydrogel was not very helpful. As shown previously, selected plants originating from xerothermic grasslands (Turnau et al. 2008) can be used for phytostabilization. Certainly these plants are able to tolerate drought and high temperature. All the plants used in the experiment at Trzebionka waste belong to a group of pseudometalophytes that usually should show the metal exclusion 18

19 strategy, comprising the avoidance of metal uptake and restriction of metal transport to the shoots. Plants used in phytostabilization should have low contents of toxic elements in shoots, to avoid contamination of the food chain. According to the presently reported data, all plant species had as high contents of Pb, Zn and As in shoots that are usually not considered as suitable for animal food. Such concentrations are, however, common in industrial areas. Still, we should try to select species that contain as little metals as possible. Among the studied species the most useful would be Melica transsilvanica, Bromus innermis, Agropyron intermedium and Anthyllis vulneraria. In those plants also, no differences in photosynthesis were shown while comparing plants from the waste with those growing on xerothermic grasslands. On the contrary, very high accumulation of potentially toxic metals was found in the case of Verbascum thapsus. The possibility to use this species in phytoextraction will be checked in future. Most plants originating from dry calcareous grasslands were successfully performing on the waste only if they were introduced as seedlings, what implies higher costs of introduction. However, these costs are still lower than in case of covering the waste with additional soil layer transported from other areas and constant watering the site. Plants from xerothermic grasslands are tolerant enough to heavy metals to survive in vegetative form and even to produce seeds. Only one species of those (V. thapsus) was able to multiply using its seeds and even more, these seeds were highly vital. For comparison, plants that are adapted to growth on wastes such as Silene vulgaris produce seeds that are able to germinate only in 45% while seeds produced in Botanical Garden germinate in 89% (Wierzbicka and Panufnik 1998). Studying the mechanisms of tolerance of V. thapsus should be the next aim of our research. Acknowledgements: We greatly acknowledge Dr. Anna Jurkiewicz (Aarhus University, DK) for the linguistic comments on this manuscript.this research based on the experiment established within the framework of the Polish Ministry of Scientific Research and Information Technology 2P04G and was carried out further under the project 197/N- COST/2008/0. References Hołynska B, Ostachowicz B, Ostachowicz J, Samek L, Wachniew P, Obidowicz A, Wobrauschek P, Streli C, Halmetschlager G, Characterisation of 210Pb dated peat core by various X-ray fluorescence techniques. Sci. Total Environ. 218, Orlowska E, Jurkiewicz A, Anielska T, Godzik B, Turnau K, Influence of different arbuscular mycorrhizal fungal (AMF) strains on heavy metal uptake by Plantago lanceolata L. Pol. Bot. Stud. 19: Strzyszcz Z, Some problems of the reclamation of waste heaps of zinc and lead ore exploitation in southern Poland. Z. Geol. Wissenschaft. 31: Turnau K, Orlowska E, Ryszka P, Zubek S, Anielska T, Gawronski S, Jurkiewicz A, Role of mycorrhizal fungi in phytoremediation and toxicity monitoring of heavy metal rich industrial wastes in Southern Poland. In Soil and Water Pollution Monitoring, Protection and Remediation. Eds. I Twardowska H E Allen and M M Häggblom. pp Springer, Dordrecht. Turnau K, Anielska T, Ryszka P, Gawronski S, Ostachowicz B, Jurkiewicz A, Establishment of arbuscular mycorrhizal plants originating from xerothermic grasslands on heavy metal rich industrial wastes new solution for waste revegetation. Plant Soil 305: Wierzbicka M, Panufnik D, The adaptation of Silene vulgaris to growth on a calamine waste heap (S. Poland). Environ. Pollution 101:

20 New outlooks in mycorrhiza applications Silvio Gianinazzi 1, Odile Huchette 2, Vivienne Gianinazzi-Pearson 1 1 UMR INRA 1088/CNRS 5184/U.Bourgogne, Plante-Microbe-Environnement, INRA-CMSE, BP 86510, Dijon Cédex, France 2 Dijon Céréales/COOPD OR R&D, INRA, BP 86510, Dijon Cedex, France silvio.gianinazzi@dijon.inra.fr Until now the application of mycorrhiza in plant production systems has focussed principally on compensating for nutrient deficiency or improving growth and productivity in presence of reduced chemical inputs (Gianinazzi & Vosatka 2004). However, tissues of arbuscular mycorrhizal (AM) plants often have higher mineral contents and several works in the past have reported enrichment in secondary flavonoid metabolites (Morandi et al. 1984, Harrison & Dixon 1994), the antioxidant activity of which is well-known. The protective effect of phytochemicals or antioxidant activities is frequently associated to health benefits of fruit or vegetables having pharmaceutical properties. With the growing interest in such aliments, researchers and producers have recently begun to address the question whether mycorrhizal plants are of better nutritional and/or health quality. For example, Khaosaad et al. (2006) and Copetta et al. (2006) reported that AM fungi can increase amounts of essential oils in basil and oregano, independent of an improved P status of plants, and we have shown increased carotene contents in sweet potato following the introduction of AM fungal inoculants under field conditions (Farmer et al. 2007). Onions are a rich source of flavonoids and sulfur-containing compounds, both of which are considered to be potentially health-promoting through their activity, for example, as antioxidants, antimicrobiotics or potential anticancer agents (Corzo-Martinez et al. 2007). In this context, Perner et al. (2008) have provided evidence that major quercetin flavonoids accumulate to a greater extent in bulbs of mycorrhizal as compared to non mycorrhizal onions. In order to further evaluate the effect of mycorrhiza on the production of beneficial compounds in onions, we have focussed studies on several health and flavour-related organosulfur compounds. For this purpose, greenhouse grown onions (cv Kador) were inoculated with either G. intraradices BEG141 or G. mosseae BEG12 in bedflats; plant growth, mycorrhizal colonisation and concentration of 2-propenyl cysteine sulfoxide (isoalliine), γ-glutamyl-s-(trans-1-propenyl)-l-cysteine (isoalliine precursor) and methyl cysteine sulfoxide were quantified. Inoculation with either AM fungus significantly enhanced the accumulation of isoalliine in onion bulbs and G. mosseae had a positive effect on methyl cysteine sulfoxide concentrations (Table 1). However, neither AM fungus affected the production of γ-glutamyl-s-(trans-1- propenyl)-l-cysteine. These effects were observed independent of plant growth which was not significantly increased in spite of high mycorrhizal colonisation of root systems. The positive effect of both AM fungi on the accumulation of isoalliine was confirmed over two years of experimentation (Figure 1). Since N supply can influence the quality and/or flavour of onions, we also evaluated mycorrhizal effects on isoalliine accumulation in the 20

21 presence of reduced N fertilisation (50%). Under these conditions, both G. intraradices and G. mosseae continued to increase isoalliine concentrations in onion bulbs (+35 to 48%). Treatment MeCSO GLUPeCS PeCSO % M Ni 1.68a 1.31a 7.56a 0a Gi 1.84a 1.06a 9.81b 85.9b Gm 3.47b 1.29a 14.16c 68.2b Table 1 : Effects of mycorrhizal inoculation (Gi, G. intraradices ; Gm, G. mosseae ; Ni, non inoculated) on the concentration (nmol/mg) of sulfur compounds in onion bulbs cv Kador : MeCSO, methyl cysteine sulfoxide ; PeCSO, 2-propenyl cysteine sulfoxide (isoalliine) ; GLUPeCs, γ-glutamyl-s-(trans-1-propenyl)-l-cysteine (isoalliine precursor). Experiment 2006, normal N fertilisation (mg/l) : Ca(NO 3 ) 2 4H 2 O, 649 ; KNO 3, 465 ; (NH 4 ) 2H 2 PO 4, 59. Different letters in columns indicate significantly different values (P=0.05) K_Ni K_Gi K_Gm Figure 1: Effects of mycorrhizal inoculation (Gi, G. intraradices ; Gm, G. mosseae ; Ni, non inoculated) on the concentration (nmol/mg) of 2-propenyl cysteine sulfoxide (isoalliine) in onion bulbs cv Kador in two independent experiments (black columns, 2005 ; white columns, 2006). Normal N fertilisation (see Table 1). These observations reinforce the evidence for a role of AM in enhancing food quality and provide a new outlook for the exploitation of this symbiosis to define systems for the production of crops with high nutritional and/or health value. The fact that this beneficial effect on food quality is expressed also at a low level of fertilizers is particularly relevant for the Mediterranean countries where agroecosystems are often fragile. References Copetta A, Lingua G, Berta G (2006) Effects of three AM fungi on growth, distribution of glandula hairs, and essential oil production in Ocimum basilicum L. var Genovese. Mycorrhiza 16: Corzo-Martinez M, Corzo N, Villamiel M (2007) Biological properties of onions and garlic. Trends food Sci Technol 18:

22 Farmer MJ, Li X, Feng, G, Zhao B, Chatagnier O, Gianinazzi S, Gianinazzi-Pearson V, van Tuinen D (2007) Molecular monitoring of field-inoculated AMF to evaluate persistence in sweet potato crops in China. Appl Soil Ecol 35: Gianinazzi S, Vosatka M (2004) Inoculum of arbuscular mycorrhizal fungi for production systems: science meets business. Can J Bot 82: Harrison MJ, Dixon RA (1994) Spatial patterns of expression and flavonoid/isoflavonoid pathway genes during interactions between roots of Medicago truncatula and the mycorrhizal fungus Glomus versiforme. Plant J 6, Khaosaad T, Vierhilig H, Nell M, Zitterl-Eglseer K, Novak J (2006) Arbuscular mycorrhiza alter the concentration of essential oils in oregano (Origanum sp., Lamiaceae). Mycorrhiza 16: Morandi D, Bailey JA, Gianinazzi-Pearson V (1984) Isoflavonoid accumulation in soytbean roots infected with vesicular-arbuscular mycorrhizal fungi. Physiol Plant Pathol 24: Perner H, Rohn S, Driemel G, Batt N, Schwarz D, Kroh LW, George E (2008) Effect of nitrogen species supply and mycorrhizal colonization on organosulfur and phenolic compounds in onion. J Agric Food 56:

23 Production of AMF s and growing needs in plant production Adholeya Alok, Reena Singh & Shanuja Beri Abstract India, which spreads over 329 million ha (hectares), has about 114 million ha of land presently under cultivation. Meeting the ever-increasing demand for food production in this second most populous country of the world is a major challenge. The land under cultivation has almost reached its saturation point with respect to productivity. This is due to the practice of intensive agriculture, which includes excessive application of fertilizers and pesticides, and introduction of modern, high yielding varieties, which generally are also highly demanding. Therefore, to further increase crop productivity, either land productivity needs to be increased or additional lands, presently under fallow or wasteland categories, need to be brought under cultivation. This can be achieved by adopting suitable technologies. Mycorrhiza technology is one such successful technology, capable of wasteland reclamation and beneficial in agriculture owing to its contribution to the plant with regard to nutritional mobilization properties. The Centre for Mycorrhizal Research, TERI, has developed the monoaxenic technology for mass production of AM (arbuscular mycorrhizal) inoculum. This technology exploits host roots genetically modified by using the bacterium Agrobacterium rhizogenes carrying Ri T-DNA plasmid to mass-produce viable, healthy, genetically pure, and high-quality fungal propagules in vitro in a sterile environment. (Figure 1). Mass production of AM fungi has been achieved with several species of genes glomous, gigaspore and_scetulospora, but G. intraradices remains the most promising, with increased spore production obtained since the early investigations on monoxenic cultivation until today. In 1992, Chabot et al. established cultures from surface sterilized spores as starter material and produced 750 spores in 30 ml medium after a period of 4 months of growth in a mono-compartmental Petri plate system. Using sheared roots as starter inoculum, Diop et al. (1994) obtained approximately 890 spores after 3 months of incubation. An advanced mode of airlift bioreactor-based production was adopted by Jolicoeur et al. (1999). These authors recovered 12,400 spores per litre of medium. St Arnaud et al. (1996) obtained 15,000 spores in a bicompartmental Petri plate in 3 4 months. This bi-compartmental system was improved by Douds (2002) by replacing the mediumin the distal compartment by fresh medium at regular intervals. With this procedure, this author obtained 65,000 spores in the distal side of the bi-compartment in a period of 7 months.with the technology developed at the Centre for Mycorrhizal Research, The Energy and Resources Institute (TERI), New-Delhi, India, the recovery of infective propagules approximated 250, ,000 spores in 3 months in 100 ml of medium (Adholeya et al. 2006). The TERI technology here adopts optimization at different levels, identifying the rate-limiting factors leading to the bulk production for commercial utilization. The AM fungi in genus Glomus provide the possibility of using colonized roots as inoculum material. This was also optimized in parallel to achieve higher root colonization, up to 70 80% (Tiwari and Adholeya 2003). The subcultivation of the 23

24 Figure 1: TERI s Mycorrhiza Technology Figure 2: Testing of mycorrhizal fertilizer with wheat Figure 3: Testing of mycorrhizal fertilizer with rice root organ and its harvest have been attained at 4 and 12 weeks respectively. Such improvement allows higher spore and propagule recovery when compared with the unit volume of media in earlier published research. This also facilitates the efficient utilization of space and energy in the production system, i.e. solid-state fermentation. Many process controls were developed in order to reduce the levels of contamination (generally from 10 15% to 3 5%, common under tropical conditions). 24

25 The technology is economical and does not require any heavy hardware and infrastructureand been transferred to five Industries. The technology has received many awards including the Biotech Product and Process Development and Commercialization Award by DBT (Department of Biotechnology, Ministry of Science and Technology, Government of India)) in 2004 and has led to the development of first-ever mycorrhizal product from in vitro based technology. The product being produced on low cost with comparatively better efficiency, found major market in North America and Europe. The technology is an innovative invention offering a partial substitute to chemical fertilizers. This provides an edge to plants to thrive better and offer enhanced yield and establishment in nutrient poor conditions. This fungal microbe, which forms a symbiotic, non-pathogenic, permanent association between the roots of land plants, is an appropriate partial substitute to mineral fertilizers and promotes yield significantly. This is extremely beneficial to almost all cultivated plants as it has a broad host range in contrast to other products available (not equivalently comparable). It is easy in application similar to chemical fertilizers. Its cost of production is highly competitive to other products and offers economic, environmental and wide spread use, advantage to farmers/growers and commercial production units. Mycorrhiza is a broad-spectrum non-specific organism. A single species is known to colonize 85% of land plants. It has a broad ecological adaptability and is known to occur in deserts as well as arctic, temperate, tropical and other inhospitable habitats. It facilitates better uptake of nutrients like phosphorus and immobile trace elements like zinc, molybdenum, etc, leading to better nutrition for plants. It offers tolerance against a range of soil stresses like heavy metal toxicity, salinity, drought, and high soil temperatures. This enhances the chances of plant survival immensely. It offers higher resistance to various soil and root-borne pathogens, thus becoming a potential disease control agent. It helps in soil conservation and soil structure stabilization, thus restoring land productivity. Mycorrhiza based product do not require to be kept at low temperature, hence provide major handling and application even with highest efficiency. Mycorrhizal fungi can utilize phosphorus from extremely low concentrations, even from unavailable sources, and provide an alternative to offset the high cost of phosphate fertilizer input. To meet the requirement of million ha of total land present in India (land under cultivation) and future land (land that can be brought under cultivation) for cultivation in a sustainable way, the mycorrhizal requirement is million tonnes annually. Few Industries in India have now initiated the production of mycorrhiza. The constructed area requirement is 5000 square feet to produce 200 tonne/annum and 12,000 square feet to produce 1000 tonne/annum Extensive field trials were conducted with successful results using mycorrhiza on different crops in different agro-climatic regions of India (Figures 2-5). The trials conducted under different programmes supported by the Ministry of Environment and Forests, the Ministry of Science and Technology in different regions across the country also met with success. These results proved the commercial viability of the technology. However, the demand for mycorrhiza requirement is too high to be met by just a few industries. 25

26 Figure 4: Testing of mycorrhizal fertilizer with potato Quality control and regulation of mycorrhizal biofertilizer In India and comparable countries, most commercial organic fertilizers are not covered by the type of national or international standards which govern the quality of chemical fertilizers. Thus, specific protocols for quality control of AM fungal inocula need to be developed and standardized for application. This is essential not only as a guarantee for producers and users but also for the protection of ecosystems. Moreover, this would also help in quality management and assessment of inoculum potential with every batch of inocula produced. Quality control of commercial AM fungal inoculum is extremely important for developing faith among the user community, along with its effectively demonstrated potentials. Unless this is achieved, the potentials will remain unexplored among the other biofertilizers. It is important to evaluate the produced inoculum from commercial units with certain reference values to ensure the strict adherence to the protocols and methodologies recommended by recognized and independent laboratories. This is most vital, as several handling errors occur at the industrial level during technology adoption and Figure 5: Testing of mycorrhizal fertilizer with Sugarcane 26

27 implementation, causing subsequent problems in product quality, which may lead to the dissatisfaction of both the end users and producers. For the mass production of AM fungi, critical benchmarks at all stages of inoculum development, covering all possible parameters desirable for ensured production, are identified. These include viability checks at processing stages until the formulation stage, ranging from the colonization of host roots, weight of dried inoculum at harvest, propagule estimations, infectivity potential of crude and formulated diluted inoculum, formulation conditions like temperature and suitable storage conditions. Such benchmarks also help institutionalized process efficiency at the production level. Once the commercial launch of the formulation is achieved, both the developer of the technology and the distributing industries share equal responsibilities for the authenticity and performance of commercialized products, and must continue to work together to evaluate responses obtained in the field by the end users. This would ensure confidence building and continuous use of these products over the years. It is important to regularly validate product performance, customer satisfaction and willingness for future use, to monitor the effectiveness of the inoculum. TERI together with Government of India recently initiated the process of standards for mycorrhiza bond product and evaluation methodologies Acknowledgements The author wish to thank financial contribution for the project from SDC, Government of Switzerland & the DBT, Government of India under the Indo-Swiss Collaboration in Biotechnology References: Diop TA, Plenchette C, Strullu DG (1994) Dual axenic culture of sheared root inocula of vesicular arbuscular mycorrhizal fungi associated with tomato roots. Mycorrhiza 5:17 22 Douds DD Jr (2002) Increased spore production by Glomus intraradices in the split-plate monoxenic culture systemby repeated harvest, gel replacement, and re-supply of glucose to the mycorrhiza. Mycorrhiza 12: Jolicoeur M, Williams RD, Chavarie C, Jortin JA, Archambault J (1999) Production of Glomus intraradices propagules, an arbuscular mycorrhizal fungus, in a airlift bioreactor. Biotechnol Bioeng 63: Tiwari P, Adholeya A (2003) Host dependent differential spread of Glomus intraradices on various Ri T-DNA transformed roots in vitro.mycol Prog 2:

28 Mycorrhizal application in Greece -the issues and future plans Reiko SHIBATA (VIORYL SA, Greece) Abstract The sensitivity of consumers about matters pertaining to food safety as well as environmental protection has increased, and resulted in the development of organic farming system in the last few years. Utilization of mycorrhizal symbioses should be considered in the system, due to reduction of application of chemical fertilizers, pesticides and irrigation. Very few scientific papers and researches are available in Greece, and it has been always difficult to find answers to critical questions concerning mycorrhizal application in the fields. However, in terms of the climate, under long dry summer and very poor winter rainfall, mycorrhizal symbioses could play an important role in the agricultural system. Since, we have few reported data, it is important initially to establish an experiment focusing on the presence or absence of mycorrhizal symbioses naturally occurring in the fields. Collecting these data will guide us to the next step which is investigating the possible benefits of applying commercial mycorrhizal inoculum. The final step will be to evaluate the outcomes of mycorrhizal symbioses in the field and create a database for future research and the development. VIORYL was established in 1946, and since the early years we have been focusing on organic farming system, developing organic insects trapping system with synthesized pheromone, bird repellents, fertilizers etc. In the last years we have launched mycorrhizal research in Greece and we are conducting experiments applying commercial mycorrhizal inoculum to fields and evaluating the results. Even though it is very early to present any data, we are expecting mycorrhizal inoculum to be a productive and reliable way achieving reduced application of chemical fertilizers, pesticides and irrigation. In this meeting, we shall present issues we are facing, future plans and challenges that lie ahead of us. 28

29 Quality Control through Record Keeping and Vouchers and a tale of confusion with Glomus intraradices. Christopher Walker, Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, UK. Different AMF have different effects on different host plants. Some have unbalanced associations, taking more from the host than they return (e.g. Modjo & Hendrix 1986; Lerat et al. 2003), sometimes depending on available substrate P concentration (Peng et al. 1993). Whole ecosystems may be affected by the identity of available AMF (Grime et al. 1987) and conversely, AMF diversity may be influenced by their ability to interact with particular hosts (Eom et al. 2000; Scheublin et al. 2007). There are edaphic and anthropogenic effects that influence AMF in different ways. As examples Glomus mosseae, may be favoured by agricultural disturbance and there are complex effects of agricultural practice on species retrieved from examination of spores produced from a variety of agricultural soils (Oehl et al. 2003) and laboratory studies have shown significant effects of different disturbance events (P or OM addition) (Boddington & Dodd 2000). In the field, AMF species recovered from soil were significantly affected by experimental changes to soil ph (Wang et al. 1985). Other AMF may require particular predispositions to germinate efficiently (Lous & Lim 1988). Acaulospora laevis from Australia requires a period of drying before the spores will germinate (Tommerup 1983). However, A. laevis from the Pacific Northwest, its type location, may never experience such an extended period of drought. The species is found in the UK and in many other lcoations in which extended dry periods are infrequent or unlikely. So perhaps species alone is not an adequate definer of at least some physiological aspects of the fungus. Similarly, if all the organisms described in the work of Croll et al. (2008) really are G. intraradices, species is an inadequate taxonomic level for defining, and thus predicting, the interaction with plants, as some individuals appear to associate predominantly with particular host species. There is evidence that competitive effects in pot culture (and therefore presumably in mass inoculum cultivation) can result in changes in AMF populations, possibly resulting in the disappearance of some species in favour of others over time. If production systems are based on open pots or beds, or even field beds, there is a high probability of contamination by other AMF or even by pathogens of plant or fungus. These factors make it important for quality control and quality assurance that the organisms present in inoculum are identified and characterised, and their interactions are understood. Good record keeping with adequate vouchers can be used to demonstrate that each batch of inoculum is free from such organisms. Such quality control can contribute greatly to assuring high quality inoculum and to prevention of transmission of deleterious organisms. At the species level, this can usually, but not always, be achieved through morphological recognition of the spores. However the existence of cryptic speciation implies that it may be necessary to use molecular tools to identify the species and individuals concerned. Depending on the complexity of the species-strain mixture, more or less effort is required. For a single species culture, a well defined species such as Scutellospora reticulata presents much less of a problem than, shall we say, a member of the complex and inadequately defined species groups such as those containing G. intraradices, G. mosseae or G. etunicatum. Mixed cultures of well characterised organisms with widely different morphologies are easier to verify than mixed cultures of very similar organisms. This is exemplified by one of the so-called G. intraradices cultures, where a supposed isolate is represented by different morphological species depending on the secondary source of the culture. Fungi identified as G. mosseae have 29

30 been shown to be widely different in their behaviour and plant growth interactions (Giovannetti et al. 2003; Lerat et al. 2003), but whether this is erroneous identification or genuine intraspecific difference remains moot. The example of G. intraradices above merits more detailed explanation. An organism from Pont Rouge, Canada with this name has been extensively reported on under two different reference numbers, DAOM (Chabot et al. 1992) and DAOM (Corradi & Sanders 2006, Alcan et al. 2006). Both numbers were listed on 19 Dec 2006 as representing the same organism in the website of GINCO-CAN, although this site is no longer active [ Examination of cultures (Fig 1) from different sources indicate that two different organisms are present under the same identifier. This is confounded by a misunderstanding of the meaning of DAOM numbers that exemplifies how a flawed documentation process can result in breakdown of quality control of all. DAOM numbers are herbarium voucher numbers. Such identities are given to samples of an organism when they are lodged in a herbarium, and represent only a snapshot of what was present in a culture at that particular time, and at that time only. They should never be used as culture identifiers. A voucher number is a historical record of what was present at a particular sampling, but cannot be used as evidence for what will be found the future. It can be used as a quality control check to verify (or otherwise) if a later subculture still has the same morphological characteristics. It may be that records are available to trace the life history of the numerous cultures of the Pont Rouge fungus (or fungi) used under these numbers, but if so, they do not seem to be in the public domain. Fig 1. Morphologically different cultures, both named Glomus intraradices, and both with the same voucher number in their ancestry. Upper from Canada via Australia. Lower, from Canada via Europe. Left, typical spores in PVLG. Right, typical spores in PVLG with Melzer's reagent. 30

31 Record Keeping A database or other record keeping system can easily be developed to solve this problem, and to provide quality assurance in respect of cultures throughout their subculturing histories. For example, the records maintained manually at the University of Western Australia for many decades provide a full and detailed account of the life histories of WUM cultures maintained at that institution, though not those of the same organisms after distribution elsewhere. A database suitable for this purpose is described here. It is based on data of origin, establishment, isolation, sampling history and culturing history. Origin For any AMF culture, an original sample must have been collected by some individual from some geographic location. The database therefore starts with this information. Geographic origin is further subdivided so that separate samples from the same location can be identified. There is confusion with the Pont Ruge cultures in this respect. One source indicates that it was collected originally by S. Parent (personal communication with S. Chabot in July 1987 from DAOM records), but the GINCO-CAN website indicated it was collected by two people C. Plenchette and V. Furlan. There is agreement that it was collected from Pont Rouge, Québec. There is agreement that it was from a Fraxinus americana woodland. Vouchers The database provides unique voucher numbers to specimens taken from the original samples, thus allowing provision of a record, with voucher specimens, of the different species or spore types present in the soil or substrate at the time of sampling. The results of any trap cultures can then be compared, along with appropriate uninoculated controls to ensure contaminant organisms have not been inadvertently established in culture. The voucher specimens numbers are linked to the origin numbers by way of the sample numbers. Vouchers are very important for quality control of cultures. Culture attempts Establishment of cultures is often first carried out by trapping in open pots with a host plant (Walker 1999), although it may be with single spores from the soil in some type of sealed system. Whichever method is chosen, the database provides unique Attempt numbers which are related in the system through sample numbers to origin numbers (and then to collector). Any vouchers that were made from the sample are recorded and linked with the attempt number for comparison at a later date. These Attempt numbers therefore are clearly distinct from, but linked to the vouchers. For the Pont Rouge cultures, I could obtain no record of original culture establishment, but it seems likely it was a soil trap. Doubtless, records do exist, but they do not appear to be in the public domain. Soil traps, of course, cannot be relied upon to provide pure cultures of a single organism. The original type culture of the species G. intraradices was trapped from a Florida citrus plantation by using root fragments and was not purified before the species was described. Subculture attempts Should the original culture attempts be successful, then further subcultures, including purification and isolation where appropriate, can be recorded in the database. Each subculture attempt receives a number, unique and linked through the database to its parent culture, and hence to all other related data in the system. The database also provides the subculture s unique parental number. It is interesting to note that the number DAOM was given in 1992, although the original culture appears to have been established in 1987 from DAOM records. I have not been able to establish the date when DAOM was issued. The 31

32 provision of linked numbers for subcultures in the database allows the ancestry of each subculture to be established, showing its relationship with other cultures, both in the past and for the future. Voucher specimens can be taken from attempts at any time and numbered, automatically linking them with their historical records. Purification Establishment of a trap or other multi-propagaule culture is a first step only. The culture must then be purified. Often this results in what is known euphemistically as a single species culture. By the nature of AMF, such a culture from root fragments or multiple spores cannot be reliably considered to be an isolate because it is likely that more than one organism is involved. Indeed, cultures of species from one genus can appear from single spore isolate attempts from a spore of a different one as smaller spores may be occupying the space provided in the lumen of larger, dead specimens. It is difficult to select spores of the same species under a dissecting microscope. Consequently, although it is possible that only a single organism has been established in culture from a multi-spore attempt, it is impossible to be absolutely certain. I have not been able to find out how or when any purification attempt was made for the Pont Rouge culture. Isolation Isolation is the separation of a fungus by single propagule into a culture that, for an asexual organism, represents an individual organism. This is normally achieved by establishing single spore cultures which of course must be maintained in contamination-free conditions (both actual an theoretical) so its identity and purity can be assured. There seems to be no evidence that the Pont Rouge culture is an isolate, though it may be. It is certainly frequently referred to as such (e.g., Croll et al. 2008). The database provides a field to enter details of whether a culture is, for example, a trap, a multi-spore attempt, or an isolation attempt. Once the culture is established as an isolate, it can be provided with a further unique number within the database that is linked to the appropriate Attempt number, and thus to its history. Conclusion Confusion such as that described above for the so-called G. intraradices from Pont Rouge can be avoided if full records are maintained and appropriate vouchers kept. A purpose-built database can be used for this, but a paper-based system or a simple system using a spreadsheet would also be adequate. Vouchers ideally should be placed in a public domain herbarium, but clearly if commercially sensitive cultures are used, they may be kept in a private collection used only by the company concerned. The following protocol is offered as a guide for record keeping and specimen preservation. 1. Origin registration Record the collector name and the geographical location of the original sample. 2. Sample identification. Detail the specific sample including, date of collection, and ecological and edaphic characteristics such as nearby AMF plants, soil ph, available P and organic matter. 3. Purification and isolation. Identify the original cultures, each one uniquely labelled. Record the details of substrate type and treatment, container size, host plant, etc. Ideally, the culture attempts should be maintained in a sealed system (e.g. Walker & Vestberg 1994). Suitable control pots need to be established to check for contamination from such problems as incomplete substrate disinfestations, carry over of contaminant spores in water or movement of propagules by insects or animals. 32

33 4. Subculturing. When the original attempt at pot culturing is successfully producing spores, use these in a process of establishing isolates. This can be directly from single spores in the first instance, or can be through one or more multi-spore cultures. Only when a single spore culture is successful should it be called an isolate. These will all be subcultures of the original isolation attempt, and a numbering system can be devised to reflect this. 5. Voucher specimens. Ideally, the original location would be sampled to establish which species are present. Samples can be taken and spores extracted, mounted on microscope slides, numbered serially, photographed, and preserved as evidence. In addition, it is useful to preserve the remains of the original sample by drying or deep-freezing in case future investigations are needed. Not all species present will be sporulating at any given time, so, for example, a soil trap may produce species that have not been seen earlier. Similarly, root fragments used for establishing cultures may yield species that were not sporulating at the time of original sampling. Once AM are established in the original traps, vouchers can be made to verify what has been found. These vouchers can include microscope slides and photographic images, but also dried or frozen subsamples for later further investigation. The original traps can be maintained until successful isolates are obtained, and then might be discarded, although ideally they would be dried or frozen in case of future needs. With good vouchers, isolation attempts can be compared to ensure that the cultures obtained correspond to the expected outcome. Differences might be because of external contamination (which will normally show up in the controls) or perhaps because apparently healthy spores were in fact dead and contained internal spores (Koske 1984) that were successful in establishing a symbiosis. Keeping these detailed records may seem tedious, but there are large gains in quality control and quality assurance to be obtained. Systems and record maintenance are at the root of such quality factors. References Alkan N, Gadkar V,Yarden O,Yoram Kapulnik Y 2006 Analysis of Quantitative Interactions between Two Species of Arbuscular Mycorrhizal Fungi, Glomus mosseae and G. intraradices, by Real-Time PCR. Applied and Environmental Microbiology 72: Boddington CL, Dodd JC 2000 The effect of agricultural practices on the development of indigenous arbuscular mycorrhizal fungi. II. Studies in experimental microcosms. Plant and Soil 218: Chabot S, Bécard G, Piché Y 1992 Life cycle of Glomus intraradix [sic] in root organ culture. Mycologia 84: Croll D, Wille L, Gamper HA, Mathimaran N, Lammers PJ, Corradi N, Eom A-H, Hartnett, DCWilson GWT Host Plant Species Effects on Arbuscular Mycorrhizal Fungal Communities in Tallgrass Prairie. Oecologia 122: Giovannetti M, Sbrana C, Strani P, Agnolucci M, Rinaudo V, Avio L 2003 Genetic Diversity of Isolates of Glomus mosseae from Different Geographic Areas Detected by Vegetative Compatibility Testing and Biochemical and Molecular Analysis. Applied and Environmental Microbiology 69: Grime JP, Mackey JML, Hillier SH, Read DJ 1987 Floristic diversity in a model system using experimental microcosms. Nature 328: Koske RE 1984 Spores of VAM fungi inside spores of VAM fungi. Mycologia 76: Lerat S, Lapointe L, Piché Y, Vierheilig H 2003 Variable carbon-sink strength of different Glomus mosseae strains colonizing barley roots Canadian Journal of Botany 81: Modjo HS, Hendrix JW 1986 The mycorrhizal fungus Glomus macrocarpum as a cause of tobacco stunt disease. Phytopathology 76:

34 Peng S, Eissenstat DM, Graham JH, Williams K, Hodge NC Growth depression in mycorrhizal citrus at High-Phosphorus supply. Plant Physiology 101: SandersIR 2008 Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices. New Phytologist 178: Scheublin TR, van Logtestijn RSP, van der Heijden MGA 2007 Presence and identity of arbuscular mycorrhizal fungi influence competitive interactions between plant species. Journal of Ecology 95: Walker C, Vestberg M 1994 A simple and inexpensive method for producing and maintaining closed pot cultures of arbuscular mycorrhizal fungi. Agricultural Science in Finland 3, Wang GM, Stribley DT, Tinker PB, Walker C 1985 Soil ph and vesicular-arbuscular mycorrhiza. pp IN British Ecological Society Special Symposium - Ecological Interactions in the soil environment: Plants, Microbes and Animals. Ed. by A. H. Fitter. Blackwell, Oxford. 34

35 AM fungi in planta : 20 years of progress in quality of cultures in vitro Strullu D.G. (1), Barbas V. (2) and Strullu-Derrien C. (1) (1) Laboratoire Mycorhizes, Université d Angers,2Bd Lavoisier, Angers Cedex, France (2) Department of genetics, University of Thessaloniki, Greece Abstract. The relationship between fungi and plants is known to be ancient; paramycorrhizas occur in the plants from the Rhynie Chert (400 million years). Eumycorrhizas have been recorded in 300 million-year-old petrified Cordaites which showed endophytic fungi in their rootlets. In modern eumycorrhizas the intraradical form comprises hyphae, arbuscules and sometimes vesicles. In 1986, Strullu and Romand achieved the first subcultivation of AM fungi, isolated from mycorrhizal roots. That result and other steps - including Agrobacterium rhizogenes transformed roots, somatic embryos, rhizogenous calli, somatic hybrids - allowed the improvement of the indefinite culture of those micro-organisms and the production of high quality inocula. These technologies require very strict controlled measures: viability, identity, purity and stability. Specific protocols for quality control of AM fungal inocula are now developed for applications. The use of in vitro cultures for inocula production appears now totally acceptable in respect to environmental and legal aspects. AM fungi inocula are now used worldwide to the satisfaction of an increasing number of plant growers and farmers. The relationship between fungi and plants is known to be ancient; paramycorrhizas occur in the plants from the Rhynie Chert (Scotland, 400 million years). The fungus Glomites rhyniensis is know from Aglaophyton sporophyte and Lyonophyton gametoppyte. Eumycorrhizas have been recorded in 300 million-year-old petrified Cordaites from Grand Croix (France) which show endophytic fungi in their rootlets. The fossil association shows intracellular hyphae within the cells and typical arbuscules with thick trunks and narrow branches (Strullu-Derrien and Strullu, 2007). In modern eumycorrhizas the intraradical form comprises intercellular hyphae, coils, arbuscules and sometimes vesicles. Bryophytes, lycopods, ferns gymnosperms and angiosperms develop arbuscular mycorrhizas. It is now recognised that the AM fungi should de placed in the Glomeromycota. Mosse and Hepper (1975) reported the first co-culture between a contaminant-free inoculum and a root organ. Strullu and Romand (1986) achieved the first subcultivation of Glomus fasciculatus, isolated from mycorrhizal roots of Fragaria and the same authors subcultivated Glomus intraradices regrowth from isolated vesicles. So the first continuous cultures of highnumber vesicle-forming Glomus species was performed. After Declerck, Seguin and Dalpé (2005) and only referring to published papers, 15 species have been cultivated with success under monoxenic conditions with production of mature spores and only 9 have been subcultivated (Figure 1). That result and other steps - including Agrobacterium rhizogenes transformed roots, somatic embryos, rhizogenous calli, somatic hybrids - allowed the improvement of the indefinite culture of those micro-organisms and the production of high quality inocula. These technologies require very strict, controlled measures to guarantee the intrinsic properties of identified strains over generations (see Declerck, Seguin and Dalpé, 2005). The viability can be confirmed through spore or vesicle germination tests. Three media could be used for these tests : SR87 medium ( Strullu and Romand, 1987) for isolated vesicles, SRM medium (Declerck et al, 1998 modified from Strullu and Romand, 1986) for spores and M medium 35

36 (Bécard and Fortin, 1988). Host are transformed or non-transformed excised roots (Daucus carota, Lycoperdicon esculentum, Allium cepa, Trifolium pratense, Medicago truncatula). Figure 1. Monoxenic cultures of AM fungi with production of mature spores and possibilities of subcultivation (after Declerck, Seguin and Dalpé, 2005) The identity of the fungal strains is mainly based on the spore morphology but the continuous cultures increase the interest of taxonomy by opening the possibility of using new material and tools as sporulation, mycelium morphology, biochemistry, molecular biology. The purity of AM fungi strains can be confirmed by starting the continuous cultures from single vesicle (Strullu and Romand, 1987; Declerck et al, 1998) or single spore ( Declerck et al, 1998). The stability of AM fungi in monoxenic cultures needs to be ascertained by long-term preservation. The situation is quite different for Acaulosopra, Gigaspora or Glomus since only the later could be cultivated over several generations. Generally there is no important variability of infectivity but some decreases of sporulation and colonisation rates can been detected. Different methods for long-term preservation are currently used.. The monoxenic culture of AM fungi with transformed roots is an excellent method for providing spores; so mass production of these spores of high quality and lower production cost has been developed. Commercial inoculum production of sterile AM fungal spores has been increasing during the last years. However, the use of spores produced from Agrobacterium rhizogenes transformed roots for the inoculation in field is sometimes restricted. As ecologists seek answers to pratical problems related to transgenic biotechnology, approaches to increase spore production using chemical methods and new methods of dual culture have been suggested. A large-scale inoculum production using rhizogenous calli is show in figure 3. 36

37 Figure 3. Schematic model of monoxenic culture of AM fungi using rhizogenous calli This method can be divided into four stages: (1) starting AM fungal material: single spores, single vesicles, mycorrhizal roots (2) plant culture tissues as somatic embryos, somatic hybrids, (3) inoculation, (4) growth of the rhizogenous calli and spore production (figure 4). The potential improvement of production performance is tested for each stage of this method (Strullu and Barbas, 2004). Figure 4. Spore production using rhizogenous calli The methodologies for in vitro cultures on root organs suffer from other limitations as the absence of photosynthetis tissues which unable the photosynthetate transfer from the plant to the AM fungus. The techniques of in vitro culture enable us to produce somatic embryos for many plant species and when these embryos are encased in a biodegradable nutritive capsule, artificial seeds are produced. Artificial seeds of alfalfa (Medicago sativa) were inoculated 37

38 with Glomus and the AM fungus regenerated from root fragments formed typical mycorrhizas with arbuscules, vesicles and spores (Strullu et al, 1989). An other autotrophic culture system in which an autotrophic plant (Solanum tuberosum) was associated to an AM fungus was developed (Voets et al, 2005). For the inoculum production, the low productivity of aseptic culture which raises the cost has been an important obstacle to putting inoculation in large-scale industrial practice. In the field of AM fungi inoculum it is necessary to check that the microorganism is present in plant, that it develops typical symbiotic structure and at least causes the plant response. Specific protocols for quality control of AM fungal inocula are now developed for horticultural and agricultural applications. Colorimetric tests, especially those using non toxic products, allow the control of the symbiotic stages. So far, few studies have examined the identity of the intraradical form in planta. An interesting objective will be to use and evaluate different molecular methods to identify the intraradical form of the AM fungi after inoculation. The associations are valuable for the plant particularly in stress conditions. The external hyphae have the ability to take up phosphate and to transport it in planta structures were it is released to the plant cells. The AM associations also increase the water uptake, this is associated with a lower transpiral flux and a better ability to extract water from substrates. The use of in vitro cultures for inocula production appears currently acceptable in respect to environmental and legal aspects. AM fungi inocula are now used worldwide to the satisfaction of an increasing number of plant growers and farmers. References Bécard G.and Fortin J.A. (1988).Early events of vesicular mycorrhizas formation in Ri-T- DNA transformed roots. New Phytol., 108, Declerck S., Seguin S. and Dalpé Y. (2005).The monoxenic culture of arbuscular mycorrhizal fungi. In : In Vitro Culture of Mycorrhizas. Editors S. Declerck, D.G. Strullu and J.A. Fortin. Springer. Soil Biology Declerck S., Strullu D.G.and Plenchette C. (1998). Monoxenic culture of the intraradical forms of Glomus sp. isolated from tropical ecosystem : a proposed methodology for germplasm collection. Mycologia, 90 (4), Mosse B. and Hepper C. (1975). Vesicular arbuscular mycorrhizal infections in root organs cultures. Physiol. Plant Pathol., 5, Strullu D.G.and Romand C. (1986). Méthode d obtention d endomycorhizes à vésicules et arbuscules en conditions axéniques. C.R. Acad. Sci., 303, Strullu D.G. and Romand C.(1987). Culture axénique de vésicules isolées à partir d endomycorhizes et ré-association in vitro à des racines de tomate. C.R. Acad. Sci., 305, Strullu D.G. and Barbas V. (2004). Method for in vitro production of mycorrhizal fungi, mycocallus and mycorrhized biological support obtained thus. PTC Application, FR2004/ Strullu D.G., Romand C., Callac P., Téoulé E. and Demarly Y. (1989). Mycorrhizal synthesis between Glomus spp. and artificial seeds of alfalfa. New Phytol. 113, Strullu-Derrien C. and Strullu D.G. (2007). Mycorrhization of fossil and living plants. C.R. Palevol., 6-7, Voets L, Dupré du Boulois H., Renard L., Strullu D.G and Declerck S. (2005). Development of an autotrophic culture system for the in vitro mycorhization of potato plantlets. FEMS Microbiology Letters, 248 (1),

39 How much diversity do we need in arbuscular mycorrhizal fungi (AMF) inoculum? Gosling P., Jones J., Bending G.D. Warwick HRI, University of Warwick, Wellesbourne, CV35 9EF, UK. Abstract Evidence for functional diversity in the arbuscular mycorrhizal fungi (AMF) is now established at all taxonomic levels, from family right down to different strains within a single species. Specific species and strains of AMF have been shown to be more or less effective at relieving plant stress from soil nutrient limitation, soil pests and disease, salinity and heavy metals. As a result, AMF diversity is important in determining the benefits gained from AMF in natural ecosystems. However, the role of AMF diversity in delivering benefits to crop plants has received less attention. We examined the effect of between 1 and 7 species of AMF on the growth of onion and clover in the glasshouse on soil from an organically managed horticultural system. Growth of onion was increased by AMF, but there was no improvement in growth by applying more than two species. Three Glomus species significantly increased growth, but neither Paraglomus occultum, Scutellospora fulgida or Acaulospora spinosa significantly increased growth. The degree of AMF colonisation was not affected by species of AMF present. Growth of clover was not influenced by application of AMF. Introduction The traditional view of arbuscular mycorrhizal fungi as generalists with wide geographical and host ranges and little functional speciality has been abandoned in the last two decades as more and more evidence of functional speciality and host specificity has emerged. The advent of molecular techniques in particular, has enabled direct investigation of which AMF species are colonising which plants, revealing complex relationships between the AMF community in the soil and that inhabiting plant roots. Increasing evidence of functional diversity and host specificity has led to the realisation that AMF diversity is likely to be important in structuring plant communities and experimental evidence supports this view (Klironomos 2003). While evidence of the importance of AMF diversity in plant communities was accumulating there was also an increasing realisation that agroecosystems are depleted in AMF, both in terms of colonisation potential and species diversity (Helgasson et al 1998). There is some limited evidence that this may lead to reduced productivity (Johnson et al 1992), though this is far from conclusive. Nevertheless, a paradigm has developed suggesting that productivity and sustainability of agroecosystems can be improved by enhancing AMF diversity, either through managing the system to enhance the native AMF community or through the introduction of new species/strains of AMF through the use of inoculum. However, there is little evidence to suggest what level of AMF diversity is required to maximise benefits or which species of AMF may be most effective. This is clearly a problem when assessing the consequences of reduced AMF diversity or deciding upon the most effective mix of AMF to apply as an inoculum. We sought to assess the degree of AMF diversity required in to gain maximum benefit to two crop plants grown in a low nutrient soil. Materials and methods We selected a sandy loam soil that had been under organic management in a horticultural rotation for 18 years. During this time it had received no animal manures or other supplementary fertilizers. As a consequence total (459 mg kg -1 ) and extractable (8.1 mg kg -1, Olsen extraction) soil P concentrations were low, other soil characteristics were; extractable K 63 mg kg -1, ph 6.10 (H 2 O), total organic carbon 2.01% and total N 0.21%. Soil was passed 39

40 thorough 3 mm sieve and irradiated with 10 k Gy radiation. Irradiated soil was moistened to 60% WHC and placed into 11cm plastic pots (753g dry wt). All pots received 10 ml of soil inoculum made from non-irradiated soil/water slurry filtered through a 38μm sieve. Pots were then inoculated with AMF. Inoculum consisted of root fragments, hyphae and spores in 50/50 silver sand/terragreen mix in which Plantago lanceolata had been grown with a single AMF species. Between zero and 7 species of AMF were inoculated into each pot with total weight of inoculum being the same for all pots (20g), divided equally between the different AMF species added. Species used are shown in Table 1. The species selection was intended to reflect the type of mixture usually found in commercial inoculum, that is, mostly Glomus species with a single species from two or three other genera. Control pots received 20 g of twice autoclaved AMF inoculum (a mixture of all 7 AMF species), plus 5ml of a 38μm filtrate of this mixed inoculum. The design was fully factorial with all possible combinations of species. The resources required to replicate such an experiment would be great and thus there was no replication except for control treatments and the treatment receiving all 7 species, which were replicated three times. Two plant species were used, white clover (Trifolium repens L.) and onion (Allium cepa L.) White clover is an important pasture species, particularly in northern Europe and its use is increasing in response to increasing fertiliser prices. Onion is a globally important vegetable crop. Within the EU production is concentrated in Mediterranean countries with Spain alone accounting for approximately one quarter of EU and 2% of world production. Both crops are strongly mycorrhizal and can be expected to respond positively to inoculation with AMF. Three pre-germinated seeds were added to each pot, which was reduced to two plants once fully established (after around one week). Pots were placed in a glasshouse and watered three times weekly to maintain soil moisture content. Plants were harvested after 15 weeks for onion and 21 weeks for clover. Roots and shoots were separated. A sub sample of roots was retained for determining colonisation using the grid line intersect method, the remaining roots and shoots were dried at 90 o C and weighed. Table 1. AMF species used Species Glomus manihotis Glomus intraradices Glomus caledonium Glomus mosseae Acaulospora spinosa Paraglomus occultum Scutellospora fulgida Culture FL879 BEG144 BEG20 BEG12 NC501 WV224 VA103B Results Growth of both onions and clover was satisfactory. By the end of the experiment bulb formation had begun in onion, though it was not very advanced, while clover had begun to flower, with around one quarter of plants flowering to some extent. Figure 1 shows onion shoot dry weight. There was no significant difference in shoot weight between zero and 1 species of AMF or between 1 and 2 species, but 2 through to 7 species all produced significantly more dry weight than zero and 3 through to 7 species produced significantly more than 1. When the effect of individual AMF species was considered alone Glomus mosseae, Glomus intraradices and Glomus caledonium produced a significant 40

41 Shoot dry wt. (g) Shoot dry wt. (g) increase in shoot weight. Glomus manihotis was the next most significant (P 0.086), none of the other species approached a significant level Number of species Figure 1. Mean shoot dry weight of onion for all species combinations. Error bars are ± 1 standard error. There were few specific species mixtures producing a significant increase in shoot weight. Of those that did they were equally likely to contain the Paraglomus, Acaulospora or Scutellospora species or just a combination of Glomus species. Root dry weight (data not shown) was not influenced by AMF (P 0.066). Total plant dry weight followed an almost identical pattern to shoot dry weight although there was a subtle difference in that total plant dry weight was significantly increased by Glomus mosseae and Glomus intraradices and Glomus manihotis, but not Glomus caledonium (P 0.081). The largest increase was with Glomus mosseae and the smallest (significant) increase was with Glomus manihotis. Examination of colonisation revealed no significant effect of AMF species or number on root length colonised. Figure 2 shows shoot dry weight of clover. The number of AMF species had no significant affect on clover shoot dry weight (P 0.427) the effect on root dry weight was similarly non significant (P 0.365). As far as individual species is concerned only G. mosseae significantly influenced growth (both shoot and root) and this was a reduction in growth. No combination of species produced a significant increase or decrease in growth Number of species Figure 2. Mean shoot dry weight of clover for all species combinations. Error bars are ± 1 standard error. 41

42 Conclusions The evidence in the literature for functional diversity in AMF and host/amf specificity is strong (Klironomos 2003, Munkvold et al 2004, Maherali and Klironomos 2007). Thus some authors have suggested that high AMF diversity is required in agroecosystems to maximise the benefits that AMF can bring. However, most intensive agroecosystems operate under low plant stress conditions and it is not clear if a diverse AMF community provides a significant benefit. Our results suggest that little benefit may be gained from high AMF diversity under conditions of low stress and that AMF may be parasitic on some plant species under these conditions. Furthermore, our results suggest that Glomus species are likely to have the largest influence of host plant growth, whether positive or negative. Although in soils with different stress factors, such as high disease pressure, this may not be the case. References Helgason T, Daniell TJ, Husband R, Fitter AH, Young JPW, Ploughing up the woodwide web? Nature 355: Johnson NC, Copeland PJ, Crookston RK, Pfleger FL, Mycorrhizae - possible explanation for yield decline with continuous corn and soybean. Agronomy Journal 84: Klironomos JN, Variation in plant response to native and exotic arbuscular mycorrhizal fungi. Ecology 84: Maherali H, Klironomos, JN, Influence of Phylogeny on fungal community assembly and ecosystem functioning. Science 316: Munkvold L, Kjoller R, Vestberg M, Rosendahl S, Jakobsen I, High functional diversity within species of arbuscular mycorrhizal fungi. New Phytologist 164:

43 Monitoring the structure and dynamics of arbuscular mycorrhizal fungus communities using Terminal Restriction Fragment Length Polymorphism of 18S rrna genes Gary D. Bending 1, Paul Gosling 1 and Giles King Salter 2 1 Warwick HRI, University of Warwick, Wellesbourne Warwick CV35 9EF, UK 2 School of Biology and Environmental Science, University College Dublin, Dublin 4Ireland Abstract Commercial AMF inocula are typically introduced into soil environments containing existing native species of AMF. Methods are needed to measure the extent to which inoculant AMF are able to successfully compete with native strains to colonise the roots of plants. Since identification of fungal species colonising plant roots is not possible using staining and morphological analysis, molecular approaches based on analysis of fungal nucleic acids offers the best way forward. PCR primers are available for amplification of specific strains, species, genera or whole communities of AMF. Since AMF inocula contain diverse strains belonging to several genera, community based profiling approaches could offer a convenient means to investigate the success of inoculant fungi. Although a number of methods are available to profile the composition of complex microbial communities, Terminal Restriction Fragment Length Polymorphism (TRFLP) of 18S rrna genes offers a cheap, high throughput approach which can provide identification and semiquantitative estimation of AMF community members. In the technique, DNA is amplified using fluorescently labelled primers. Amplicons are digested using restriction enzymes and the size and quantity of Terminal Restriction Fragments (TRF) determined on a sequencer using capillary electrophoresis. TRF can be related to specific organisms by reference to TRF produced from sequenced clones derived from the sample or strains described in DNA databases. In the current paper we describe application of the 18S rrna-trflp technique to investigate the structure and dynamics of AMF communities in agricultural systems. The influence of soil phosphorus (P) status on AMF associated with maize and soybean was studied in a field experiment at Wellesbourne. A P gradient was established and maintained by application of phosphate fertiliser to an arable field to provide UK soil phosphorus indices between 1 and 8 (between 10 and 200 mg available P kg -1 ). Five years following establishment of the gradient, crops of maize and soybean were grown. Roots of each crop were taken at pre-flowering, flowering and just prior to harvest. DNA was extracted from roots and TRFLP performed using the primers AML1 and AML2, which amplify AMF with the exception of the Archaeosporales. It was shown that the total number of TRF was affected by plant species, with more TRF, and hence higher diversity, in soybean relative to maize. Number of TRF was unaffected by sampling time. Elevated soil P status reduced number of TRF in soybean by up to 80 %, but had no effect on number of TRF associated with maize roots. Multi Dimensional Scaling (MDS) showed that the structure of communities was significantly affected by crop type but not sampling time. The structure of AMF communities at the highest P level was significantly different to that at lower P levels for both crops. Cloning demonstrated that the dominant members of the community were Glomus caledonium, G. etunicatum, G. sinuosum and G. mosseae. Restriction analysis of the cloned sequences revealed that the G. caledonium community consisted of 4 clone types, each of 43

44 which gave different TRF, while the G. etunicatum community consisted of 2 clone types, each of which gave distinct TRF. Relating the individual clone types to TRFLP profiles enabled the response of the different species, and for G. caledonium and G. etunicatum, the different clone types, to crop type and soil P status to be determined. The applicability of 18S rrna TRFLP to differentiate between AMF found in inocula and native communities will be discussed. 44

45 Characterization of Mediterranean AM: initiation of a novel functional marker approach Vicente C. 2 and Arnholdt-Schmitt B. 1* Corresponding author: * eu_chair@uevora.pt 1 EU Marie Curie Chair, ICAM, University of Évora, Portugal 2 Instituto Nacional de Recursos Biológicos L-INIA Elvas (Ex-Estação de Melhoramento de Plantas) Estrada Gil Vaz Apartado Elvas, Portugal Introduction The Mediterranean climate, predominant in the Mediterranean basin, is characterized by cold winters and warm summers with an enormous irregularity in the distribution of rainfall in space and time. In accordance with summer drought, this system can be divided into three regions: an arid region to the south; semi-arid regions in the eastern littorals, the western Spanish coast and large islands; and a humid region to the north (Makhzoumi, 1999). The complex interactions between rainfall and temperature, together with geology, topography, soil and vegetation cover, account for the Mediterranean ecosystems fragility and susceptibility to degradation (Makhzoumi, 1999). The intensification of desertification conditions, rather by natural or anthropogenic processes, imposes the search for new alternatives for soil rehabilitation and recuperation of vegetation diversity. The application of arbuscular fungi (AMF) in agricultural systems comprehends mainly in the recuperation of poor and degraded soils and in the establishment of new practices for the recovering of plant populations. Besides the well-known advantage of phosphorus uptake, AM fungi can also benefit plants by: increased shoot and root biomass; increased uptake of other nutrients (nitrogen and cooper), limited uptake of toxic heavy metals, increased resistance to pathogens; improved defence against or altered interactions with herbivores; and one of the most ambitious and controversial profit, improved plant-water relations (Newsham et al., 1995). Applications of AMF in Mediterranean ecosystems AMF in the recovering of desertified Mediterranean Ecosystems Several studies have been developed in attempt to decrease the pressure of desert-like conditions along the Mediterranean systems, emphasizing the importance of mycorrhizal symbioses in the rehabilitation/restoration of degraded ecosystems. Herrera et al. (1993), Requena et al. (1996) as well as Azcón-Aguillar et al. (2003) highlighted the importance of natural mycorrhizal potential associated with woody species from the natural succession in semiarid ecosystems. In addition, Alguacil et al. (2005) proved that the establishment of mycorrhizal shrub species favours the reactivation of soil microbial activity, which is linked to an increase of aggregates stability and an acceleration of nutrient cycles. Concerning AMF diversity in the Mediterranean regions, Ferrol et al. (2004) analysed one of the most representative shrub species, Pistacea lenticus, which is a also target plant for re-vegetation programmes. The author found that diversity was confined especially to Glomus (G. constrictum, G. viscosum, G. claroideum, G. mosseae) and Paraglomus occultum, typical species from Mediterranean systems except P. occultum. 45

46 AMF in important agronomic cultures of Mediterranean ecosystems Here we focus on two important Mediterranean species: the emblematic Olea europea L and Cicer arietinum L. AM inoculation is known to increase survival rate and development of micro-propagated plantlets (Azcón-Aguilar and Barea, 1997). AM application is thus important for the establishment of young olive trees. Santos-Antunes (2002) reported that the inoculation of olive plantlets just before field sowing, can produce higher rates of development in the field and also better chances to survive to an eventual drought period or attack by pathogenic agent. Assessing the effectiveness of native fungal isolates as inoculants of olive trees, Calvente and co-workers (2004) found that AM fungi diversity in the rhizosphere was considerably low under the monoculture practice and that the degree of responsiveness to AM inoculation not only varied depending on olive genotype but also on the AM fungi inoculated. The pulse crop Cicer arietinum L. (chickpea), as the third most important food legume globally, plays an important role in the maintenance of soil fertility and for human/animal consumption with optimal nutritional compositions (high protein content with a good percentage of true protein digestibility) (Saxena, 1990). Chickpea productivity can be affected by different biotic (such as pathogenic agents) and abiotic factors (water and nutrient deficiencies), in which AM fungi inoculation may help to overcome. Rao et al. (2006) reported that a dual inoculation of Rhizobium sp. and Glomus fasciculatium has increased the nodulation, nitrogen and phosphorus concentration in plants and yield of chickpea. However, care must be taken in view of the results of Weber et al. (1993). These authors observed a negative effect of AM inoculation related to the reproductive growth. Mycorrhiza plants had improved P uptake and vegetative growth, but failed to achieve high productivity probably because of the terminal drought effects under Mediterranean climate conditions. Jalali and Thareja (1981) observed the suppression of Fusarium wilt incidence in chickpea and concluded that mycorrhizal roots were nutritionally healthier than non-mycorrhizal roots. Jalali (1992) also reported that the presence of AM fungi in chickpea exerted a significant impact on the spectrum of root exudation as well as plant mineral nutrition. Gahoonia et al. (2006) underlined the need to study root traits as support for breeding drought-tolerant and nutrient efficient high yielding varieties. These authors suggested that the combination of root traits found in chickpea (high acid exudation, greater root-hair density and length) could enhance the crop adaptation to marginal and dry areas. Presentation of a novel functional approach for AM characterization For the application of arbuscular mycorrhizal fungi in agricultural systems the following parameters will be critical: 1. survival of the inoculum in the natural habitat 2. competitiveness with other soil organisms in the rhizosphere 3. maintenance of the genetic integrity 4. efficiency of the AMF in terms of a. inducibility of branching b. symbiotic effectiveness to improve availability of nutrients and water and provide pathogen tolerance All these parameters can potentially be influenced by the genetics of the AM fungi. Genetic techniques are available to characterize the genomic background of AM fungi (Franken and Requena, 2001; Ferrol et al., 2004; Hause and Fester, 2005, Turrini et al. 2008). Typically, AMF isolates are characterized by methodologies using SSR, SSU or ITS identification. However, single spores can carry several genetic variants (Helgason and Fitter, 2005). Large 46

47 spores contain typically hundreds of nuclei and high degrees of polymorphism make this task a difficult one (Young, 2008). Van der Heijden and Scheublin (2007) point to the importance of considering the functional diversity of AMF. The composition of AMF can have important impact on plant performance, plant community structure and ecosystem functioning. The authors stress a missing link between knowledge on the functional significance of AMF and ecosystem functioning. Recently, it started that selected genes, such as phosphate transporters, have been characterized in AM fungi (e.g. Isayenkov et al. 2004). This approach aims a specific characteristic related to the symbiotic efficiency. We will propose working on the alternative oxidase (AOX) as a marker to characterized biodiversity/gene diversity and at the same time the functional integrity of AMF communities. Tamasloukht et al. (2003) speculated on the role of alternative respiration in the pre-symbiotic phase to enable survival of AM fungi spores and AOX is the key enzyme in this pathway. This candidate gene could serve to monitor most of the critical parameters for application. AOX belongs to an ancient gene family that is distributed in all kingdoms of life (McDonald, 2008). During evolution, fungi AOX has developed separately from plants. Characterization in mycorrhiza plants will be possible. The authors of this paper are working on AOX characterization in diverse plant species and want to initiate research on the hypothesis that regulation of AOX from plants and AM fungi is connected during the pre-symbiotic phase and plays a crucial role in mycorrhiza colonisation (Arnholdt-Schmitt, 2008). References Arnholdt-Schmitt B, A novel gene candidate of socio-economic interest? Proceedings of COST 870 meeting:from production to application of arbuscular mycorrhizal fungi in agricultural systems: a multidisciplinary approach, p. 47. Azcón-Aguilar C, Palenzuela P, Roldán A, Bautista S, Vallejo R, and Barea JM, Analysis of the mycorrhizal potential in the rhizosphere of representative plant species from desertification-threatened Mediterranean shrublands. Applied Soil Ecology 22:29-37 Calvente R, Cano C, Ferrol N, Azcón-Aguilar, and Barea JM, Analysing natural diversity of arbuscular mycorrhizal fungi in olive tree (Olea europea L.) plantations and assessment of effectiveness of native fungal isolates as inoculants for commercial cultivars of olive plantlets. Applied Soil Ecology 26: Ferrol N, Calvente R, Cano C, Barea JM, Azcón-Aguilar C, Analysing arbuscular mycorrhizal fungus Diversity in shrub-associated resource islands from a desertificationthreatened semiarid Mediterranean ecosistema. Applied Soil Ecology 25: Ferrol N, Azcón-Aguilar C, Bargo B, Franken P, Gollotte A, González-Guerrero M, Harrier LA, Lanfranco L, van Tuinen D, and Gianinazzi-Pearson V, Genomics of arbuscular mycorrhizal fungi. Applied mycology and biotechnology 4: Franken P, and Requena N, Analysis of gene expressionin arbuscular mycorrhizas: new approacheas and challenges. New Phytologist 150: Gahoonia TS, Rawshan A, Malhotra RS, Jahoor A, Matiur Rahman M, Root morphological and physiological traits and nutrient uptake of chickpea genotypes. Journal of Plant Nutrition (accepted). Hause B, and Fester T, Molecular and cel biology of arbuscular mycorrhizal symbiosis. Planta 221: Herrera MA, Salamanca CP, and Barea JM, Inoculation of Woody legumes with selected arbuscular mycorrhizal fungi and rhizobia to recover dersertified Mediterranean ecosystems. Applied and Environmental Microbiology 59: Helgason T, and Fitter A, The ecology and evolution of the arbuscular mycorrhizal fungi. Mycology 19:

48 Isayenkov S, Fester T, and Hause B Rapid determination of fungal colonization and arbuscule formation in roots of Medicago truncatula using real-time (RT) PCR. J. Plant Physiol. 161: Jalali BL, and Thareja ML, 1981, Suppression of Fusarium wilt of chickpea in vesiculararbuscular mycorrhizal inoculated soils. International Chickpea Newsletter 4: Jalali BL, and Chand H, Chickpea wilt. In: Plant Diseases of International Importance. Vol. 1. Diseases of Cereals and Pulses (U.S. Singh, A.N. Mukhopadhayay, J. Kumar, H.S. Chaube, ed.), Prentice Hall, Englewood Cliffs, NJ, USA, McDonald A E, Alternative oxidase: an inter-kingdom perspective on the function and regulation of this broadly distributed cyanide-resistant terminal oxidase.functional Plant Biology 35: Makhzoumi J, The Mediterranean context. In: Makhzoumi, J. and Pungetti, G (edts). Ecological Landscape Design and Planning: The Mediterranean context. GBR: Spon Press. pp Newsham KK, Fitter AH, and Watkinson AR, Multi-functionality and biodiversity in arbuscular mycorrhizas, Tree 10: Requena N, Jeffries P. and Barea AM, Assessment of natural mycorrhizal potential in a desertified semiarid ecosystem. Applied and Envirornmental Microbiology 62: Santos-Antunes AF, 2002, As Micorrizas e o crescimento de plantas: o caso da oliveira. Melhoramento 38: Van der Heijden MGA, and Scheublin TR, Functional traits in mycorrhizal ecology: their use for predicting the impact of arbuscular mycorrhizal fungal communities on plant growth and ecosystem functioning. New phytologist 174: Weber E, Saxena MC, George E, and Marschner H, Effect of vesicular-arbuscular mycorrhizaon vegetative growth and harvest index of chickpea grown in northern Syria. Field Crops Research 32: Young JPW, The genetic diversity of intraterrestrial aliens. New Phytologist 178: 465 Tamasloukht MB, Séjalon-Delmas N, Kluever A, Jauneau A, Roux C, Bécard G, and Franken P, Root factors induce mitochondrial-related gene expression and fungal respiration during the development switch from asymbiosis to presymbiosis in the arbuscular mycorrhizal fungus Gigaspora rosea. Plant Physiology 131: Turrini A, Avio L, Bedini S, and Giovannetti M, In situ collection of endangered arbuscular mychorrhizal fungi in a Mediterranean UNESCO Biosphere Reserve. Biodiversity and Conservation 17:

49 Morphological and molecular characterization of some arbuscular mycorrhizal fungi, potential candidates to use in the protection of dune plants of the Mediterranean Sea Błaszkowski J., Gábor M. Kovács G. M. Department of Plant Protection, University of Agriculture Slowackiego 17, PL Szczecin, Poland Department of Plant Anatomy, Institute of Biology, Eötvös Loránd University, Pázmány Péter sétány 1/C, 1117 Budapest, Hungary Arbuscular mycorrhizal fungi of the phylum Glomeromycota are considered to belong to the most common soil fungi in the world and associate with at least 80% of vascular land plants (Smith and Read 1997). Sites harbouring exceptionally abundant and diverse populations of arbuscular fungi are maritime sand dunes, mainly because of their low nutrient content and organic components (Dalpé 1989, Koske 1987, 1988, Nicolson and Johnston 1979, Tadych and Błaszkowski 2000). Of many plants of dune sites, Ammophila arenaria (L.) Link is the most important sand-fixing species in maritime dunes of Europe (Rodríguez-Echeverría and Freitas 2006), and its roots commonly host arbuscular mycorrhizae and diverse populations of spores of the Glomeromycota (Kowalchuk et al 2002, Nicolson and Johnston 1979, Tadych and Błaszkowski 2000). Other dune plants, including many protected and threatened species, generally also coexist with diverse spore populations of these fungi. The association of arbuscular mycorrhizal fungi with maritime dune plants may be of considerable ecological significance for their establishment and growth, because these fungi enhance plant nutrient uptake, increase plant tolerance to drought and salt stress, and protect them against soil pathogens and nematodes (Koske et al. 2004). At least 32 newly described species of arbuscular mycorrhizal fungi have originally been associated with roots of dune plants and many others have occurred in maritime dunes (Błaszkowski 2003; Sridhar and Beena 2001). Of the almost 70% of the described species of the Glomeromycota found by the first author in dune sites of the Mediterranean Sea located in, e. g., Portugal, Spain, France, Italy, Greece, Turkey, Israel, Cyprus and northern Africa, the fungi most frequently occurring in the field conditions and easily and abundantly sporulating in both pot trap and one-species-cultures were Archaeospora trappei, A. delicata, A. lacunosa, A. paulinae, A. scrobiculata, A. spinosa, Glomus aggregatum, G. aurantium, G. drummondii, G. constrictum, G. corymbiforme, G. eburneum, G. fasciculatum, G. irregulare, G. intraradices, G. lamellosum, G. pustulatum, G. trimurales, G. versiforme, G. walkeri, G. xanthium, Scutellospora calospora, S. fulgida, S. pellucida, and S. persica. Their wide distribution and easy cultivation in greenhouse conditions indicate them to be the best candidates to use in production of inoculum assigned for the preservation of protected and threatened dune plant species. During the presentation, the most important diagnostic morphological and molecular properties of some of the species listed above will be shown. The species will be also compared with those with which they may easily be confused. Additionally, the steps of formulation of an inoculum and its incorporation in the field conditions will be proposed and discussed. 49

50 References Błaszkowski J, Arbuscular mycorrhizal fungi (Glomeromycota), Endogone, and Complexipes species deposited in the Department of Plant Pathology, University of Agriculture in Szczecin, Poland. Koske RE, Gemma JN, Corkidi L, Sigüenza C, Rinkón E, Arbuscular mycorrhizas in coastal dunes. In: M. I. Martínez, N. P. Psuty (eds.). Coastal dunes, ecology and conservation. Ecol. Studies 171: Kowalchuk GA, De Souza FA, Van Veen JA, Community analysis of arbuscular mycorrhizal fungi associated with Ammophila arenaria in Duch coastal sand dunes. Molecular Ecology 11: Nicolson TH, Johnston C, Mycorrhiza in Gramineae. III. Glomus fasciculatum as the endophyte of pioneer grasses in maritime sand dunes. Trans. Br. Mycol. Soc. 72: Rodríguez-Echeverría S, Freitas H, Diversity of AMF associated with Ammophila arenaria spp. arundinacea in Portuguese sand dunes. Mycorrhiza 16: Sridhar KR, Beena KR, Arbuscular mycorrhizal research in coastal sand dunes: a review. Proc. Nat. Acad. Sci. India. 71: Tadych M, Błaszkowski J, Arbuscular fungi and mycorrhizae (Glomales) of the Slowiński National Park, Poland. Mycotaxon 74:

51 The real-time quantitative PCR assay: from its detection of fungi pathogenic on trees to its use detecting AMF Luchi N., Capretti P. Department of Agricultural Biotechnology, Plant Pathology Section, University of Florence. Piazzale delle Cascine 28, I Florence, Italy Abstract Molecular techniques have been particularly useful to detect specific micro-organisms in different substrates (i.e. wood, soil, water) without the need to isolate a pure culture. Molecular techniques are more reliable than the traditional methods, which have several limitations in that they are difficult to standardise, usually do not give quantitative results (O Connel et al., 1998; Loric et al., 1995) and tend to be cumbersome and time consuming, especially if hundreds of samples have to be analysed (Simon et al., 1992). In the case of fungal communities that colonise plants, some species may be difficult to isolate, while other species are masked by endophytic micro-organisms that overgrow them on agar media (Catal et al., 2001). Over the last few years the use of PCR techniques has greatly increased and expanded, including new PCR approaches such as real time quantitative PCR (rt-qpcr), which has proved to be a useful tool to quantify amplification products. The main feature of rt-qpcr is that the PCR products are detected at each cycle, in real-time, allowing them to be quantified during the exponential phase of the run. This is unlike classical PCR, where quantification is carried out only at the end-point (or plateau phase) of the run (Heid et al., 1996), so that their accuracy is lower. The sensitivity of rt-qpcr is mainly due to a different chemistry, which uses fluorogenic molecules that release fluorescence during PCR synthesis. Some fluorogenic molecules are single molecules (such as for example SYBR green) that intercalate in a double stranded DNA, but some are also labelled oligonucleotides that bind with a specific region of the DNA target (Bustin, 2000). Rt-qPCR has been applied in plant pathology to detect fungal pathogens and for other purposes. In this field rt-qpcr has furthered our understanding of plant pathogen interactions (Schena et al., 2004). An interesting feature that has been found is that some fungi, including pathogenic fungi, have a latent phase in standing trees, when they live in those trees but do not cause any symptoms. When these trees become debilitated for some reason, however (mostly by drought) the fungus starts to grow, and to colonize and damage the host-tissue. Diagnostic methods based on DNA may be able to detect infections with such fungi at an early stage. During the last few years, real-time PCR has therefore been developed to detect fungi in apparently healthy plant tissue, in three different pathosystems: Diplodia pinea on Pinus nigra (Maresi et al., 2007), Biscogniauxia mediterranea on Quercus cerris, Q. ilex (Luchi et al., 2005) and Biscogniauxia nummularia on Fagus sylvatica (Luchi et al., 2006). The ABI Prism 7700 Sequence Detection System with the TaqMan TM assay has proved to be a rapid and sensitive means to quantify amplification products in real-time PCR. This system uses a dual-labelled fluorogenic probe that anneals between the two primers. The probe contains two fluorescent dyes covalently linked at its ends: at the 5 end there is a reporter [FAM (6-carboxy-fluorescein)] and at the 3 end there is a quencher [TAMRA (6-carboxytetramethyl-rhodamine)]. Reporter and quencher are excited by laser during PCR. After a 51

52 denaturation step, the fluorogenic probe hybridises to the template DNA within the region defined by the forward and reverse primers. During the polymerisation steps, the 5-3 exonuclease activity of the Taq-polimerase cleaves the probe (Holland et al., 1991). The fluorescent signal from the free reporters, detected during PCR thermocycling, allows quantification of the template copies. Computerised systems such as the ABI Prism 7700 Sequence Detection System make it possible to analyse a great number of samples at a time (up to 96 per run) and to reduce processing times quite substantially (to about 2 hours). Higher initial costs are offset by the greater number of samples processed. Research on forest tree pathogens showed that with this system lower amounts of fungal DNA (less than 10 pg) could still be detected in healthy shoots. The technique was sensitive enough to detect fungal DNA from an ascospore suspension (Luchi et al., 2005). The data showed that the rt-qpcr detected low quantities of amplified DNA. The studies confirmed the reliability of this technique for tree diseases and suggested that it could also be used on samples collected from natural populations. Sensitive molecular techniques such as rt-qpcr, make it possible to identify a specific organism in a DNA mixture consisting of plant tissue and other micro-organisms that usually live into the host. It is also possible to monitor harmful fungal parasites, particularly those that have a long latency phases without showing any symptoms, such as fungal endophytes. In forest pathology, when monitoring fungal diseases, this molecular approach gives useful information before the diseases are clearly evident in the landscape, and it can also be utilised in probing the relationship between disease and environmental parameters, which may promote fungal spread. So far there have been only a few studies dealing with the application of rt-qpcr to arbuscular mycorrizal fungi (AMF).The rt-qpcr technique can be used to test: a) AMF inoculum; b) AMF in the soil; c) the interaction between AMF and the host. a) AMF inoculum. It is important to develop a standardised control system to analyse the inoculum. Conventional methods, such as MPN, only have a limited capacity to estimate the number of inoculum spores (Baar, 2007). The rt-qpcr assay could determine the quality and the quantity of the fungal inoculum. The assay has already been used reliably to determine the spore dilution of B. mediterranea (Luchi et al., 2005) and G. mossae (Bohm et al., 1999). The rt-qpcr assay also requires only minute quantities of starting material, reducing loss of inoculum. b) AMF in the soil. The amount of AMF in the soil may be affected by cultural practices and by soil management, with negative consequences on the abundance and diversity of the AMF. The rt-qpcr technique can be useful to monitor changes in the AMF community (Cavagnaro et al., 2007). c) AMF in planta. The most common tool conventionally used to detect AMF in host tissue is light microscopy. However, this method does not distinguish between fungal species or quantify their presence at species level. As for fungal pathogens, rt-qpcr is particularly useful to monitor the colonisation process of fungi in plants. A recent study has shown an interesting application of rt-qpcr to detect different AMF in host plants (Alkan et al., 2006). This method could be utilised to study gene-expression in the plant microbe interaction. Liu et al. (2007) has shown that it might be possible to apply rt-qpcr to study gene expression as result of AM symbiosis. The understanding of gene expression could be improved using a sophisticated technique named Laser Microdissection Pressure Catapulting (LMPC), which dissects out single cell types from microscopic tissue sections (Day et al., 2005). LMPC uses an inverted microscope and a nitrogen laser in order to microdissect out selected cells that are subsequently levitated 52

53 (catapulted) into a collection cap using the same laser source. The cells so collected are then used to extract RNA and DNA, and for downstream applications such as expression studies or gene copy number evaluation using real-time PCR (Pinzani et al., 2006). We optimised the LMPC technique and combined it with real-time RT-PCR in order to detect -tubulin genes in the bark tissue of Norway spruce (Picea abies). This method is an important tool to study gene modulation at the level of specific cells, and can be used to exploit the interaction between plant cells with micro-organisms such as AMF. The sensitivity and specificity of rt-qpcr represents one of the most notable aspects of this innovative technique, which has proved to be highly efficient because of the great number of samples that can be analysed in a single run. For the applications mentioned above, real-time PCR is a promising technique to study the biology of AMF. This new technique may also have implications for AM fungi, and may facilitate monitoring these micro-organisms in a wide range of substrates References Alkan N, Gadkar V, Yarden O, Kapulnik Y, Analysis of quantitative interactions between two species of arbuscular mycorrhizal fungi, Glomus mosseae and G. intraradices, by real-time PCR. Applied and Environmental Microbiology, Baar J, Innovative thoughts for the development of a quality control system. In: Proceedings of the COST Action 870. Contribution of mycorrhizal fungi to agro-ecosystems: how to bridge the gap from small-scale production to commercial utilization May Budapest, Hungary Bohm J, Hahn A, Schubert R, Bahnweg G, Adler N, Nechwatal J, Oehlmann R, Osswald W, Real-time quantitative PCR: DNA determination in isolated spores of the mycorrhizal fungus Glomus mosseae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants. Journal of Phytopathology 147, Bustin SA, Absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology 25: Catal M, Adams GC, Chastagner GA, Detection, identification and quantification of latent needlecast pathogens and endophytes in symptomless conifer foliage by PCR and Dotblot assays. In: Forest Research Institute Res. Papers. Proceedings of the IUFRO Working Party Shoot and foliage Diseases, Hyytiälä, Finland, Cavagnaro TR, Jackson LE, Scow KM, Hristova KR, Effects of arbuscular mycorrhizas on ammonia oxidizing bacteria in an organic farm soil. Microbial Ecology 54: Day RC, Grossniklaus U, Macknight RC (2005) Be more specific! Laser microdissection of plant cells. Trends Plant Sci 10: Heid CA, Stevens J, Livak KJ, Williams PM, 1996.Real time quantitative PCR. Genome Research 6: Holland PM, Abramson RD, Watson R, Gelfand DH, Detection of specific polymerase chain Reaction product by utilizing the 5' to 3' exonuclease activity of Thermus acquaticus. DNA polymerase. Proceedings of the National Academy of Sciences of the United States of America. 88: Liu J, Maldonado-Mendoza I, Lopez-Meyer M, Cheung F, Town CD, Harrison MJ, Arbuscular mycorrhizal symbiosis is accompanied by local and systemic alterations in gene expression and an increase in disease resistance in the shoots. Plant Journal 50: Loric S, Dumas F, Eschwege P, Blanchet P, Benoit G, Jardin A, Lacour B, Enhanced detection of hematogenous circulating prostatic cells in patients with prostate adenocarcinoma by using nested reverse transcription polymerase chain reaction assay based on prostatespecific membrane antigen. Clinical Chemistry 41:

54 Luchi N, Capretti P, Pinzani P, Orlando C, Pazzagli M, Real time PCR detection of Biscogniauxia mediterranea in symptomless oak tissue. Letters in Applied Microbiology 41: Luchi N, Capretti P, Vettraino AM, Vannini A, Pinzani P, Pazzagli M, Early detection of Biscogniauxia nummularia in symptomless European beech (Fagus sylvatica L.) by TaqMan real-time PCR. Letters in Applied Microbiology 43: Maresi G, Luchi N, Pinzani P, Pazzagli M, Capretti P, Detection of Diplodia pinea in asymptomatic pine shoots and its relation to the Normalized Insolation index. Forest Pathology 37: O'Connell CD, Juhasz A, Kuo C, Reeder DJ, Hoon DSB, Detection of tyrosinase mrna in melanoma by reverse transcription-pcr and electrochemiluminescence. Clinical Chemistry 44: Pinzani P, Orlando C, Pazzagli M 2006a. Laser-assisted microdissection for real-time PCR sample preparation. Molecular Aspects of Medicine. 27: Schena L, Nigro F, Ippolito A, Gallitelli D, Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi. European Journal of Plant Pathology 110: Simon L, Levesque L, Lalonde RCM, Rapid quantitation by PCR of endomycorrhizal fungi colonizing roots. PCR Methods and Applications 2:

55 Efficacy and persistance of introduced AM fungi in rehabilitation processes. Estaún V, Busquets M, Calvet C. Camprubí A. IRTA Investigació i tecnologia agroalimentàries. Protecció Vegetal. Cabrils. Cra de Cabrils Km 2, Cabrils (Barcelona) Spain Introduction In semiarid climates the establishment of a plant cover is the most important step in the restoration of degraded areas, such as quarries or urban wastelands, to avoid further degradation and desertification.. Under Mediterranean conditions the restoration becomes difficult because of the many constraints associated with the climate (dry periods followed by torrential rains) and the soil (shallow, stony soils with low organic matter and high ph). In most instances the plants used in these projects are grown under supraoptimal conditions and do not present any mycorrhizal symbiosis. The success level of these revegetations is low (Sort & Alcañiz, 1996). The use of organic fertilizers such as sewage sludge has increased the success of the plants survival (Sort & Alcañiz, 1996), however it has as a drawback considering the development of many volunteer plants that are nonmycorrhizal and extremely aggressive and can delay the targeted species growth and development, besides added problems of soil and water contamination. These problems have prevented the spread of this practice in sensitive areas. Mycorrhiza are reported to reduce the detrimental effects on plant growth of soilassociated stresses such as lack of nutrients, high ph and climate associated stresses such as drought and high temperatures (Requena et al, 2001; Caravaca et al, 2003; Alguacil et al, 2005), however the effect of the symbiosis under field conditions is sparsely documented and with diverse results (Clemente et al, 2005). The establishment of a plant cover can be done transplanting shrubs and /or trees or casting seed in the areas to be revegetated. Both strategies are routinely used, depending on the accessibility of the area to be reclaimed and on economic issues. Some rehabilitation projects contemplate the use of a nurse crop of herbaceous species to stop the erosion process and improve the soil for future rehabilitation activities (Skousen and Zipper, 1996). In this paper we present the results of pretransplant inoculation with AMF on the revegetation of two quarries and one urban wasteland using native shrubs and the establishment of an herbaceous crop directly sown with a hydroseeder as a first rehabilitation step. The urban small open space was included in the experiment due to the growing importance of these areas in reclamation projects. Materials and methods Quarry revegetation The first quarry studied is located in MontRal (Tarragona, Spain; 41º 16 N 1º07 E). The quarry has been mined for several decades for the extraction of stone blocks for ornamental use. The experimental area covered a terrace of m 2 formed by stocked limestone gravel and other debris. Topsoil originally retrieved from the area to allow the excavation and stored in piles was spread over the terrace to provide a substrate for revegetation. (Photo1) 55

56 Photo 1: Revegetation of the quarry with added topsoil The second quarry studied is located in Castellar del Vallés (Barcelona Spain, 41º 36 N 2º03 E). The quarry has been mined extensively to obtain ground stone for building and production of cement. The experimental area was a dumping area with unusable ground rock debris with no added topsoil. (Photo2) Photo 2: Revegetation of the quarry with no added topsoil The plants used for the experiment were: Lavandula angustifolia Mill. and Santolina chamecyparissus L. for both quarries with Juniperus phoeniceae L. and Thymus vulgaris L., for the first quarry and Anthyllis citisoides and Rosmarinus officinalis L. for the second quarry The AM inoculum used was a mixture of roots and rhizosphere substrate of leek plants inoculated with Glomus intraradices Schenk &Smith BEG72 and grown in Terragreen. The growth parameters evolution was estimated as plant volume for L. angustifolia, T. vulgaris, S. chamaecyparissus, R. officinalis and A. cytisoides and as plant height for J. phoenicia. To assess mycorrhizal colonisation composite samples were taken from rhizosphere soil with a soil core borer in three points chosen at random for each of the repeated treatments of 6 plants, samples were observed under a binocular microscope to evaluate mycorrhizal colonization (Koske and Gema,1989; Giovanetti and Mosse, 1980) To assess the diversity and persistence of the introduced AM fungus in the area after 23 months growth, eight 1cm root pieces of each of the root composite samples (inoculated and noninoculated) were used for DNA extraction using a chelex extraction method (van Tuinen et al. 1998, Kjøller & Rosendahl 2000). Primary PCR was performed with the eukaryote specific primers LSU0061 (LR1) and LSU0599 (NDL22) followed by nested PCR with the primer combinations LSURK4f and LSURK7r (van Tuinen et al. 1998, Kjøller & Rosendahl 2000) and FLR3 and FLR4 (Gollotte et al. 2004). PCR were performed as described by Kjøller & Rosendahl (2000). All positive PCR were sequenced using RK4f or FLR3 as sequencing primer. Parsimony analyses were conducted in PAUP. Urban open space revegetation The small urban space is located in Badalona (Barcelona Spain, 41º26 N 2º13 E), it is a small layout, in a slope, between blocks of apartments, underutilised and unattractive. The soil was heavily degraded and contained building debris from nearby construction sites. The plants used for this experiment were Rosmarinus officinalis L. inoculated with AMF or not in the nursery. 56

57 Hydroseeding experiment The hydroseeding system was used to improve soil coverage and erosion control in the quarry where no topsoil was added and in a small waste land in an urban location. The plants used were the legumes Medicago lupulina and Lotus corniculatus and the grasses Lolium perenne, Festuca ovina and Brachypodium. phoenicoides. The fungus, G. intraradices, was also chosen according to the results of a previous experiment (Estaún et al, 2006). The results of the hydroseeding procedure were evaluated in the field and in greenhouse conditions. Three months after the hydroseeding procedure, the number of grasses and legumes in five squares of 20cm x 20cm in each of the replicated treatments in the field locations was recorded. Plants under greenhouse conditions were harvested and the shoot weight of legumes and grasses was recorded separately, the root weight/cm 2 and the root colonisation were determined. Results and Discussion Quarry revegetation All plants established the mycorrhizal symbiosis at the nursery. At transplant inoculated and noninoculated plants were similar in size. After 8 months growth in the quarry with added top soil all control plants sampled presented the symbiosis and there were no differences in plant growth (Photo 3) Molecular probes show that the most widespread fungi were G. intraradices, and Glomus microaggregatum although other fungi were present in the roots of the control plants(figure1a). Figure 1a: Diversity of AM fungi detected in mycorrhizal roots of noninoculated and inoculated shrubs 23 months after transplant in the quarry with added topsoil In the quarry where no top soil was added all inoculated plants grew better, although 60% of the non inoculated plants sampled presented the symbiosis. Glomus intraradices was the only fungus detected in the roots of the inoculated plants whilst in the roots of the control plants other fungi were found (Figure 1b). Figure 1b: Diversity of AM fungi detected in mycorrhizal roots of noninoculated and inoculated shrubs 23 months after transplant in the quarry with no added topsoil 57

58 Our results show that adding stored top soil is a good system to enhance mycorrhizal colonisation in quarry restoration; otherwise it is important to inoculate the plants used with an effective fungus. Molecular probes show that in the Mediterranean ecosystems studied, with eroded soils and high ph, the AMF species diversity is very low and G. intraradices is present in a high percentage of the samples, indicating the resilience and adaptation to these conditions of this species. Urban open space revegetation As many of the areas that need rehabilitation often are in pronounced slopes, the growth and development of Rosmarinus officinalis plants inoculated and noninoculated with G. intraradices was evaluated in different gradient slopes. The inoculation at the nursery level was found to increase plant growth and coverage in the two gradients of slope considered (Figure 2 ). Figure 2: Height of R. officinalis plants grown in 20º and 40º slopes inoculated with G. intraradices (M) or noninoculated (C) 12 months after trasplant. For the rehabilitation of the urban layout, R. officinalis was considered to be the most adequate plant due to its soil and climate stress resistance and its aromatic and ornamental properties. Nursery inoculated plants survived transplant and the summer drought stress (Figures 3a and 3b) (Photo 4). The inoculation of plants under these circumstances is cost effective due to the high costs of labour if plants need to be replaced. Figure 3: Survival (a) and growth (b) of R. officinalis plants grown in a urban layout inoculated with G. intraradices (M) or noninoculated (C) 8 months after trasplant. 58

59 Photo 4: Revegetation of a urban layout with R. officinalis 8 months after plant establishment (A. Plants inoculated with G. intraradices, B: plants noninoculated) Hydroseeding experiment The addition of inocula increased the total weight of plants recovered both in the greenhouse and in the field experiments (Figure 6) (Photo5). The same results apply for the analysis of the legumes dry weight, where the weight of legumes in the mycorrhizal treatments was increased by tenfold respect to the nonmycorrhizal treatments. Considering the dry weight of grasses there is no significant effect of the inoculation with G. intraradices although there is a significant effect of the inoculation in the legumes/grasses ratio, which is higher in all mycorrhizal treatments. Figure 4: Total number of plants and numbers of legumes and grasses in two locations (A and B) three months after the hydroseed application. Data are means of the plants observed in five 20cm x 20 cm squares in each of the three replicates per treatments per location ± 1.98SE of total number of plants 59

60 Photo 5: Hydroseeding of a urban layout 3 months after application (A: noninoculated, B: inoculated with G. intraradices) Conclusions Transplanting native plants inoculated with AMF is a good system to establish shrubs and trees in eroded semiarid lands (Requena et al,2001; Caravaca et al, 2003; Alguacil et al, 2005) but it is impracticable when considering large areas (Greipsson and ElMayas 1999). The integration of the AMF inoculation with the hydroseeding technique might permit the use of mixtures of native grasses and legumes, and can facilitate the establishment of other mycorrhizal plants (Enkhuya et al, 2005) in large or inaccessible areas where transplanting cannot be considered as an option for rehabilitation. References Alguacil MM, Caravaca F, Roldán A (2005). Changes in the rhizosphere microbial activity mediated by native or allochtonous AM fungi in the reafforestation of a Mediterranean degraded site. Biology and Fertility of Soils 41, 5968 Caravaca F, Barea JM, Palenzuela J, Figueroa D, Alguacil MM, Roldán A (2003). Establishment of shrub species in a degraded semiarid site after inoculation with native or allochtonous arbuscular mycorrhizal fungi. Applied Soil Ecology 22, Clemente AS, Werner C, Máguas C, Cabral MS, MartinsLouçao MA, Correia O (2004). Restoration of a limestone quarry: effect of soil amendments in the establishment of native Mediterranean sclerophyllous shrubs. Restoration Ecology 12, 2028 Estaún V, Vicente S, Calvet C, Camprubí A, Busquets M (2007). Integration of arbuscular mycorrhizal inoculation in hydroseeding technology. Effects on plant growth and interspecies competition. Land Degradation and Development 18, Gollote A, van Tuinen D, Atkinson D (2004). Diversity of arbuscular mycorrhizal fungi colonising roots of grass species Agrostis capillaries and Lolium perenne in a field experiment. Mycorrhiza 14: Giovannetti M, Mosse B (1980). An evaluation of techniques for measuring vesiculararbuscular mycorrhizal infection in roots. New Phytologist 87: Kjoller R, Rosendahl S (2000). Detection of arbuscular mycorrhizal fungi (Glomales) in roots by nested PCR (polymerase chain reaction) and SSCP (single stranded conformation polymorphism) Plant and Soil 226, Koske RE, Gemma JN (1989). A modified procedure for staining roots to detect VA mycorrhizas. Mycological Research 92,

61 Requena N, PérezSolis E, AzcónAguilar C, Jeffries P, Barea JM (2001). Management of indigenous plantmicrobe symbiosis aids restoration of desertified ecosystems. Applied and Environmental Microbiology 67, Rosendahl S, Stukenbrock E (2004) Community structure of arbuscular mycorrhizal fungi in undisturbed vegetation revealed by analysis of LSU rdna sequences. Molecular Ecology 6, Sort X, Alcañiz JM (1996). Contribution of sewage sludge to erosion control in the rehabilitation of quarries. Land Degradation and Development 7, 6976 Enkhtuya B, Poschl M, Vosatka M Native grass facilitates mycorrhizal colonisation and P uptake of tree seedlings in two anthropogenic substrates. Water Air and Soil Pollution 166: Greipsson S, ElMayas H Large scale reclamation of barren lands in Iceland by aerial seeding. Land Degradation and Development 10: Skousen J, Zipper CE Revegetation species and practices. Reclamation Guidelines for Surface Mined Lands in Southwest Virginia. Powell River Project. Viginia Cooperative Extension Publication

62 Effects of inoculation with two AM fungi on two poplar clones grown at high zinc concentration. Guido Lingua a, Valeria Todeschini a, Cinzia Franchin b, Stefano Castiglione c, Stefania Biondi b, Patrizia Torrigiani b, Giovanni D Agostino d, Graziella Berta a a Dipartimento di Scienze dell Ambiente e della Vita, Universita` del Piemonte Orientale Amedeo Avogadro,Via Bellini 25/G, I Alessandria, Italy - guido.lingua@mfn.unipmn.it b Dipartimento di Biologia evoluzionistica sperimentale, Universita` di Bologna, Via Irnerio 42, I Bologna, Italy c Dipartimento di Chimica, Università di Salerno, Stecca 7, Via Ponte don Melillo, I Fisciano (SA), Italy. d Istituto di Virologia Vegetale del CNR, Strada delle Cacce 73, I Torino, Italy. Abstract Since 2003, a wide study has been conducted on the use of poplar, associated or not to AM fungi, for phytoremediation purposes. Part of the research was conducted in field, working a site polluted with copper and zinc (over 1000 ppm in the first layers of soil Castiglione et al. submitted manuscript), made available by KME Italy, part in glasshouse (Todeschini et al. 2007; Todeschini et al., submitted manuscript; Lingua et al., 2008), under more controlled conditions, and part in vitro, under laboratory conditions (Castiglione et al., 2007; Franchin et al., 2007). The glasshouse experiments were carried out on non-sterile soil (in order to simulate field conditions), supplemented or not with zinc chloride (300 mg/kg of soil), using two registered, commercial clones of poplar: Populus alba Villafranca (VF) and Populus nigra Jean Pourtet (JP), inoculated or not with an arbuscular mycorrhizal fungus, either Glomus mosseae or G. intraradices (Lingua et al. 2008). After about six months of growth, plants were harvested and analysed for their morphological parameters, zinc concentration in their organs (root stem leaves), leaf polyamine concentration (putrescine, spermine and spermidine) and for leaf morphology and ultrastructure. The applied zinc concentration resulted to be toxic for plants, as shown by their morphological parameters: growth was inhibited in comparison to control (no metal) plants in both poplar clones (Fig. 1). Spontaneously occurring mycorrhizal colonization was prevented (VF) or strongly inhibited (JP) by the metal, but pre-inoculation resulted in colonization levels comparable with those of plants grown without metal supplementation. It is worth nothing, however, that arbuscule abundance was severely reduced by zinc pollution (Fig. 2). Zinc was differentially accumulated in the two poplar clones. In general, VF accumulated larger amounts of metal than JP. In both clones, zinc concentration was highest in the leaves, followed by roots and shoots. Differences between the two clones were mostly evident in the leaves, where VF accumulated over 2000 mg of zinc per kg of dry weight, while JP reached about 1500 mg/kg. Mycorrhizal colonization slightly (but significantly) reduced zinc concentration in the leaves of VF plants and in the shoots of JP poplars. The two poplar clones differentially responded to zinc in pre-inoculated plants. JP plants did not show any effect of the fungus, while VF poplars, when inoculated with G. mosseae but 62

63 not with G. intraradices, resulted to be similar, as far as size and morphology are concerned, to control (no metal) plants, suggesting a protective effect of the symbiosis (Fig. 1). These results are associated to variations in the profile of polyamine expression. In VF leaves, where the highest amount of zinc was accumulated, the concentration of free putrescine was greatly increased by the metal treatment, in comparison with the controls, while the concentration of conjugated putrescine was drastically decreased. Pre-inoculated plants, in the presence of zinc, did not show such a response and their free and conjugated putrescine concentrations were not different from those of the controls, in spite of a leaf zinc concentration of about 1900 mg/kg (Fig. 3). Fig. 1 Shoot dry weight of P. alba Villafranca (left) and of P. nigra Jean Pourtet (right). NoMet: plants not supplemented with zinc; Zn: plants supplemented with zinc; ZnGm: plants treated with zinc and pre-inoculated with G. mosseae; ZnGi: plants treated with zinc and preinoculated with G. intraradices. Bars represent standard errors. Different letters indicate significant differences ( p< 0.05). Other morphological parameters showed similar trends. From Lingua et al., Fig. 2. Mycorrhizal colonization (M%, white columns) and arbuscule abundance in the colonized area (a%, black columns) in the root system of P. alba Villafranca (a) or of P. nigra Jean Pourtet (b). NoMet: plants not supplemented with metal; Zn: plants supplemented with zinc; ZnGm: plants treated with zinc and pre-inoculated with G. mosseae; ZnGi: plants treated with zinc and pre-inoculated with G. intraradices. Bars represent standard errors. Different letters indicate significant differences ( p < 0.05). From Lingua et al.,

64 Fig. 3 - Mean concentration and standard error (bars) of free (white columns) and soluble conjugated (black columns) putrescine in leaves of P. alba Villafranca (left) and of P. nigra Jean Pourtet (right), treated or not with zinc, and pre-inoculated or not with G. mosseae or G. intraradices. NoMet: plants not supplemented with zinc; Zn: plants supplemented with zinc; ZnGm: plants treated with zinc and pre-inoculated with G. mosseae; ZnGi: plants treated with zinc and pre-inoculated with G. intraradices. Bars represent standard errors. Different letters indicate significant differences ( p < 0.05). Spermidine and spermine did not show similar variations. From Lingua et al., The observed effects, cannot be described as nutritional effects, as the phosphate concentration in the soil and in the plants was rather high and also because of the reduced amounts of arbuscules. Therefore, it is likely that the arbuscular mycorrhizal fungus G. mosseae could promote some response in the plant, increasing its tolerance to zinc. Microscopic analyses, carried out on VF leaves, with light transmission, TEM and SEM instruments, provided additional information on the effects of zinc on the leaves and on the modification induced by AM colonization. The thickness of the leaf lamina (and of each leaf tissue: palisade parenchyma and spongy tissue) was increased in the presence of zinc, but this effect was reduced in zinc treated, G. mosseae-inoculated plants, and completely reverted in zinc treated and G. intraradices-inoculated plants. Sections of the palisade parenchyma, parallel to the leaf surface, showed much larger intracellular spaces and detachment of the cell walls, following zinc treatments (Fig. 4). These effects were not observed in G. mosseae inoculated plants, but in all cases zinc accumulation mainly occurred in the cell walls, as shown by a histo-chemical staining (Fig. 5). Considering the ultrastructural level, some modifications were induced by the treatment with zinc. In the first place, a large number of crystals could be observed in the parenchyma cells close to the vascular bundles (Fig. 6). Such crystals were recognized to be calcium oxalates combining raman spectroscopy and SEM EDS analyses. Second, primary starch in the chloroplast was very abundant in the controls, but not in the metal-treated plants. Both these effects were recovered in zinc treated plants, pre-inoculated with G. mosseae (Fig. 7). The increased presence of calcium oxalate crystals might be connected with the larger intracellular space and with the cell wall localization of zinc. Indeed, zinc is a stronger ligand than calcium and could displace it from the cell wall, where it is usually present. Cells strictly 64

65 control calcium concentration; increased levels of calcium could be made physiologically and osmotically inactive producing oxalate crystals. The present data suggest an important role for AM fungi in increasing the plant tolerance to metals, with relevant consequences for phytoremediation applications. However, they also stress the importance of the right plant-fungus combination: VF poplars, more sensitive to zinc pollution, most likely because of a larger metal accumulation, highly benefited of the inoculation with G. mosseae, but not with G. intraradices. On the other hand, JP plants did not show relevant effects of the mycorrhizal symbiosis. In addition, it is noteworthy that several effects of AM colonization could be detected in the leaves, a district of the plant that is not directly interested by the presence of the fungus. Such effects concerned anatomy, cell wall organization, photosynthesis and sugar metabolism. Finally, a model is proposed for the disruption of tissue organization observed in the palisade parenchyma, where the presence of zinc, associated to the displacement of calcium (and hence to the formation of calcium oxalate crystals) and to the variation in the concentration of free and conjugated putrescine, could be considered responsible of the observed effects. Fig. 4 Section of VF poplar leaves, parallel to the surface, showing the palisade parenchyma of control (left) and zinc treated plants. (right) Note the distance between the cell walls on the right. Fig. 5 (left) Cross section of a leaf of VF poplar, zinc treated, stained with dithizone, in order to show the localization of zinc. 65

66 Fig. 7 TEM images of leaves of VF poplars, showing details of chloroplasts. From left to right: control, zinc-trated, zinc + G. mosseae, and zinc + G. intraradices. References Castiglione S, Franchin C, Fossati T, Lingua G, Torrigiani P, Biondi S, High zinc concentrations reduce rooting capacity and alter metallothionein gene expression in white poplar (Populus alba cv. Villafranca). Chemosphere 67: Castiglione S, Todeschini V, Franchin C, Torrigiani P, Gastaldi D, Cicatelli A, Rinaudo C, Berta G, Biondi S, Lingua G, submitted manuscript. Screening of a large poplar clone collection for phytoremediation of heavy metal-contaminated soil: a field trial. Franchin C, Fossati T, Pasquini E, Lingua G, Castiglione S, Torrigiani P, Biondi S, High concentrations of Zn and Cu induce differential polyamine responses in micropropagated poplar (Populus alba L. cv. Villafranca). Physiologia Plantarum 130: Lingua G, Franchin C, Todeschini V, Castiglione S, Biondi S, Burlando B, Parravicini V, Torrigiani P, Berta G, Arbuscular mycorrhizal fungi differentially affect the response to high zinc concentrations of two registered poplar clones, Villafranca (Populus alba L.) and Jean Pourtet (Populus nigra L.). Environmental Pollution 153: Todeschini V, Franchin C, Castiglione S, Burlando B, Biondi S, Torrigiani P, Berta G, Lingua G, Responses of two registered poplar clones to copper, after inoculation, or not, with arbuscular mycorrhizal fungi. Caryologia 60: Todeschini V, D Agostino G, Boccaleri E, Roccotiello E, Bonelli G, Berta G, Lingua G, submitted manuscript. Leaf modifications induced by zinc accumulation in leaves of poplar inoculated or not with arbuscular mycorrhizal fungi. 66