En respuesta a la falta de suelo fértil e incontaminado, se están introduciendo masivamente en agricultura los denominados SUSTRATOS, que son muy utilizados especialmente en los semilleros y viveros, es decir, en instalaciones en las que se propagan plantas a partir de semillas y se cultivan hasta alcanzar un tamaño óptimo para su trasplante al campo o invernadero. Muchos de los sustratos utilizados actualmente están basados en turba, lo cual contribuye a agravar otro problema medioambiental, pues las turberas de donde se extrae son ecosistemas de alto valor ecológico que deben ser preservadas. Desde los años 90, se ha experimentado para intentar encontrar sustratos no basados en turba y que puedan suponer una alternativa viable al uso de la misma. Por otro lado, y pese a conocerse los efectos nocivos de los productos químicos sobre la calidad del suelo, la enorme dependencia que tienen los agricultores actuales de utilizar productos químicos como pesticidas o fertilizantes no puede ser abandonada sin disponer de alternativas viables que no supongan pérdidas de rendimiento para el agricultor. Por todo ello es necesario desarrollar nuevos bioproductos que no tengan los inconvenientes medioambientales de los tratamientos químicos actuales pero que al mismo tiempo tengan una efectividad comparable como pesticidas o fertilizantes.

THE EFFECTS OF TRICHODERMA

Trichoderma is a beneficial fungus naturally present in most of the soil. Its antagonistic action against soil borne pathogens results in very effective suppression those pathogens. This action as biopesticide is the most commonly known of Trichoderma fungi. However, some Trichoderma families have other secondary beneficial effects of great importance for the nearby plants.

ACTION AS BIO-FERTILISER AND BIO-STIMULATOR

Some strains of Th have also fertiliser and stimulator effects, as the organic matter degraded by the fungi can be absorbed as nutrients by the nearby plants. Moreover, it’s been proven that Th fungi produce phyto-hormone-like substances that have a beneficial effect in the growth of the plants around. For example, the application of Trichoderma harzianum and Pseudomonas fluorescens led to increases in dry matter content, starch, total soluble sugars (TSS) and reducing Sugar contents in leaves of sunflower (Lamba et al. 2008). A resistance mechanism induced in plants has been suggested to be the stimulatory effect of Trichoderma on the enzymes of anitoxidative reaction, e.g. peroxidase activity, as it was shown for date palms (e.g. Ben Khaled et al. 2008).

INDIRECT EFFECT OF PROTECTION OF THE PLANT IN FROM OF SOME AERIAL PARTHOGENS

Very recent investigations lead by Dr. Pascual (R&D Manager of Microgaia) in collaboration with CSIC-CEBAS have shown that Trichoderma can also have an indirect effect of protection of the plant in front of some aerial pathogens (and not only in front of soil pathogens as it was always thought). (Martinez Medina et al., 2009, 2010). In short, the mechanism for creating this protection is based in the fact that plants have evolved a sophisticated immune system to “perceive” attack by alien organisms, and to translate this “perception” into an appropriate level of defense. In this case, Trichoderma is perceived as the alien organism and the plant improves its immune system. The plant immune system is based on a complex signaling network that is highly flexible in its capacity to recognize and respond to the invader encountered, which involve a number of signalling molecules (jasmonic and salicylic routes), that can move around the plant. It is well known that Trichoderma can induce this molecule synthesis; therefore a primary immune system can be produced by Trichoderma inoculation.

SCHEME SHOWING THE EXTENSION OF THE PLANT ROOT SYSTEM CAUSED BY THE AMF SYMBIOSIS

Arbuscular mycorrhizal fungi inhabit the ecosystems of terrestrial plants as a natural way to acquire nutrients from soils (Pirozynski and Dalpe 1992). The plant response to AMF depends on numerous factors including the genotype, soil-related and climate properties, and the agrochemical inputs into the cultivation system. Mycorrhiza’s problem-solving potential applies to:

• increasing agronomic production
• enhancing C sequestration in terrestrial ecosystems to stabilize the atmospheric CO2 concentration.
• converting degraded, polluted or desertified soils to restored land or sustainable agro-ecosystems.
• developing sustainable farming/cropping systems aimed at improving water use efficiency and soil properties to combat increasing erosion and minimize risks of water pollution and eutrophication.

Thus, AM fungi are regarded as essential components of sustainable soil–plant systems (Schreiner et al. 2003). As an ecological biofertilizer, a bioprotectant against environmental stresses, such as drought or nutrient deficit, an agent controlling root pathogens (Phytophtora, Fusarium, Pythium) and a soil improver acting as an anti-erosion agent, mycorrhiza possesses great potential in sustainable or organic agriculture. Moreover, it appears that its contribution to carbon sequestration is substantial; mycorrhizal fungi can be an important soil carbon sink and often constitute 20–30% of total soil microbial biomass (Leake et al. 2004).

Extensive mycorrhizal research conducted during the last few decades has focused on the mechanisms of the symbiosis and its role in the management of sustainable crop production, where mycorrhiza could play an important role in combating these problems (Vosátka and Albrechtová 2008). An example of an already successfully developed technology is the EUREKA project E!3375 Mycotagrif: organic or semiorganic cultivation of blueberry plants, with the participation of Symbio-m.

Another beneficial effect is that AMF induce plant physiological changes that affect the quality and safety of food crops, including a higher production of antioxidants or essential oils, and reduced uptake of pollutants such as heavy metals to plant tissues (Toussaint 2007; Toussaint et al. 2007; Vosa ́tka and Albrechtova ́ 2008, Geneva et al. 2010). Although no clear mechanism other than an improvement in the nutritional status (mainly P) has been proposed (Toussaint 2007), yet the beneficial fungus–plant interactions has shown enhancement in productivity of crops by synthesizing an increased level of active compounds (Rai et al. 2001). For example, the suitable selection of host plant–fungus genotype led to an altered accumulation of essential oil levels in AM-colonized plants of Mentha arvensis (Freitas et al. 2004) and sweet basil (Copetta et al. 2006, 2007; Toussaint et al. 2007). Thus, the use of mycorrhiza is being extended to herbal plants in order to get maximum benefits by the steadily increasing herbal medicine industry (Ernst 2000).

Recent research shows also an increase in the antioxidant properties of harvested plants, which increases the biological value of mycorrhizal fungi in agricultural and horticultural production. For example, the application of microbial consortia (Pseudomonas + Azotobacter + Azosprillum + AMF) has an incredible effect on lycopene, antioxidant activity and potassium contents on tomato (Ordookhani et al. 2010). Tolerance to Fusarium wilt and anthracnose and changes in antioxidative abilities in AMF strawberry plants have also been observed in strawberry plants inoculated with AMF (Li et al., 2010).

Experiments have also been conducted to prove the efficiency of AMF as a bioprotector agent against different pathogens, such as its effects against wilt induced by Verticillium spp. in pepper (Goicoechea et al, 2010) or against Fusarium in asparagus (Barea et al, 2010).

The Czech company Symbiom, one of the project partners, is specialist in the production of mycorrizal products used to supply the plant with much more extra water and nutrients, contributing to superior growth, resistance and health of many crops.

THE USE OF AFM-TH DUALLY INOCULATED COMPOST AS SUBSTRATE

Compost can be used as organic amendment to further increase soil fertility or control some pests (Pascual et al., 2002; Ros et al., 2005; Alabouvette et al., 2006) or as a growing medium in horticulture (Bustamante et al., 2008; López-Mondéjar et al., 2010). The main advantages of compost use are:

• The high content in organic matter and nutrients.
• It is pathogens free.
• Its suppressive effect against phytopathogens (Ros et al., 2005; Segarra et al., 2007).

Most of the compost posses some capacity to increase disease suppression in horticultural crops and improve plant health (Deepak et al., 2008; Ros et al., 2004; Borrero et al., 2004), but they have showed different efficacy depending on their specific biotic and abiotic characteristics (Ros et al., 2004; Albouvette et al., 2006). For this reason, they are inoculated with suppressive microorganisms such as Trichoderma sp. to improve its suppressive efficacy, facilitating microorganism survival and the reuse of waste materials (Lopez-Mondejar et al., 2010). Species within the genus Trichoderma are some of the most widely-utilized fungal biological control agents in agriculture (Papavizas, 1985; Chet, 1987). The antagonistic effect by Trichoderma sp. has been demonstrated against Fusarium, Phytophthora, Sclerotinia, Rhizoctonia and Pythium (Inbar et al., 1996; Schoeman et al., 1999; Aerts et al., 2002; Rojo et al., 2007; Almeida et al., 2007).

These fungi act as mycoparasites against competing fungi by secreting hydrolytic enzymes such as chitinase and glucanase, which break down cell walls (Kubicek et al., 2001). Trichoderma sp. also produces antibiotic compounds which influence the biocontrol capacity (Howell, 2003). Their rapid growth allows these species to directly compete for space and nutrients with phytopathogens (Sivan et al., 1989; Hjeljord & Tronsmo, 1998), while also indirectly fighting infection through stimulating plant growth and inducing acquired resistance mechanisms in the plant (Bailey and Lumsden, 1998).

The mycoparasitic capacity of Trichoderma genera depends on the specie and in particular on the specific isolate (Markovich & Kononova, 2003). Isolates of T. harzianum with a high potential for the secretion of hydrolytic enzymes can be obtained naturally from compost and/or agricultural soil (Rincon et al., 2008). The strain Th78, isolated and registered by Microgaia Biotech (using RNA combined with quantitative real-time PCR techniques to specifically monitoring the strain and ensuring its amount and activity), has extraordinary properties as biopesticide, biofertiliser and biostimulator in several crops, and has shown great results when applied to substrates to be used in plant nurseries or soilless agriculture. Th78 is also characterized by its high survival capacity in different organic materials (BernalVicente et al., 2009). Specifically for the Gaia substrate, the Th78 concentration was reduced only 10 times 20 days after its inoculation, while the same concentration was reduced by 3 orders of magnitude in peat substrate after the same period of 20 days.

Melon plants that have been infected with F. oxysporum through rhizosphere, treated with a commercial liquid Trichoderma strain and treated with Th78 under a solid matrix.

The effect of Th78 and the different AMFs used, and their interactions, on the plant immune system has been evaluated by Dr. Pascual research group at CEBAS-CSIC (Martinez-Medina et el., 2009, 2010), where different signaling compounds have been detected depending on the presence of a specific fungi or their interactions (the plant immune system is based on a complex signaling network to recognize and respond to the invader encountered).

The inoculation of compost with suppressive microorganisms such as Trichoderma sp. improves the suppressive efficacy of composts, decreasing the disease and enhancing the plant health (López-Mondéjar et al., 2010). Once inoculated in the GAIA compost, the strain T78 has been demonstrated to show a high biopesticide effect against a range of plant pathogens such as Fusarium oxysporum, Rhizoctonia solani, Pythium ultimum, etc., and further it has also been immobilised into compost that synergistically improves the effects of Th and the compost separately (Spanish Pending Patent of Microgaia Biotech).

COMPLEMENTARY TECHNICAL INFORMATION ABOUT COMPOST GAIA INOCULATED WITH TRICHODERMA HARZARINUM T78

The use of specific compost can be an attractive approach to reduce plant pathogens. They can be used as organic amendment that further to increase soil fertility, they can also control some pests (Pascual et al., 2002; Ros et al., 2005; Alabouvette et al., 2006;) or as a growing medium in horticulture (Bustamante et al., 2008; López-Mondéjar et al., 2010). The main advantages of compost use are the: 1) high content in organic matter and nutrients, 2) pathogens free, and 3) suppressive effect against phytopathogens (Ros et al., 2005; Segarra et al., 2007). Most of the compost posses some capacity to increase disease suppression in horticultural crops and improve plant health (Deepak et al., 2008; Ros et al., 2004; Borrero et al., 2004), but they have showed different efficacy depending on their specific biotic and abiotic characteristics (Ros et al., 2004; Albouvette et al., 2006). For this reason, if they are inoculated with suppressive microorganisms such Trichoderma sp. to improve its suppressive efficacy, facilitating microorganism survival and reusing waste materials (Lopez-Mondejar et al., 2010).

Species within the genus Trichoderma are some of the most widely-utilized fungal biological control agents in agriculture (Papavizas, 1985; Chet, 1987). The antagonistic effect by Trichoderma sp. has been demonstrated against a range of agriculturally-devastating phytopathogenic microorganisms, including those within the genus Fusarium (pathogen used in this study), Phytophthora, Sclerotinia, Rhizoctonia and Pythium (Inbar et al., 1996; Schoeman et al., 1999; Aerts et al., 2002; Rojo et al., 2007; Almeida et al., 2007).

These fungi act as mycoparasites against competing fungi by secreting hydrolytic enzymes such as chitinase and glucanase, which break down cell walls (Kubicek et al., 2001). Trichoderma sp. also produces antibiotic compounds which influence the biocontrol capacity (Howell, 2003). Their rapid growth allows these species to directly compete for space and nutrients with phytopathogens (Sivan et al., 1989; Hjeljord & Tronsmo, 1998) while also indirectly fighting infection through stimulating plant growth and inducing acquired resistance mechanisms in the plant (Bailey and Lumsden, 1998).

The mycoparasitic capacity of Trichoderma genera depends on the specie and in particular for the specific isolate (Markovich & Kononova, 2003). Isolates of T. harzianum with a high potential for the secretion of hydrolytic enzymes can be obtained naturally from compost and/or agricultural soil (Rincon et al., 2008) as it was selected the natural strain Trichoderma harzianum T78, that was isolated from a compost from a huge amount of different locations. It was characterized by its high capacity to survive in different organic materials (BernalVicente et al., 2009) and further its high recognized mycoparasitism action than permits to use it as biopesticide in a huge range of plant pathogens. Trichoderma suppress various disease-causing fungal pathogens, such as Fusarium sp. (e.g. Martinez-Medina et al. 2009, Martinez-Medina et al. 2010, Srivastava et al. 2010), Rhizoctonia solani (e.g. Chandanie et al. 2009), Colletotrichum (e.g. Zivkovic et al. 2010) Botrytis (e.g. Mehofer et al. 2009, rev. By Kohl 2009).

Furthermore, it is known that plants have evolved a sophisticated immune system to “perceive” attack by alien organisms, and to translate this “perception” into an appropriate level of defense. The plant immune system is based on a complex signaling network that is highly flexible in its capacity to recognize and respond to the invader encountered, which involve a number of signaling molecules, including the action of the phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (Kunkel and Brooks 2002). Their signaling pathways differentially regulate defenses that are effective against specific types of attackers. Pathogens that require a living host (biotrophs) are commonly more sensitive to SA-dependent defense responses, whereas pathogens that kill the host and feed on the content (necrotrophs) and herbivorous insects are generally affected by JA- and ET-dependent defences. Interestingly, hormone-regulated defense pathways intimately cross-communicate in an antagonistic or synergistic manner, providing plants with a powerful regulatory capacity to quickly adapt to their biotic and abiotic environment and to utilize their resources in a cost-efficient manner (10). In some of the last results recently published by Dr Pascual research group at CEBAS-CSIC (MartinezMedina et al., 2009, 2010). it has been demonstrate the effect of Trichoderma harzianum T78 or the different AMFs used, and their interactions, on the plant different immune system, where different signalling compounds have been detected depending on the presence of a specific fungi or the interactions.

Specificity of Th78 in comparison with the rest of analysed Trichoderma strains once it is inoculated in GAIA compost. The strain T78 have been demonstrated to show a high biopesticida against a range of plant pathogens such as Fusarium oxysporum, Rhizoctonia solani, Pythium ultimum, etc., and further it also have been immobilised into compost that will improve synergistically the effects of Trichoderma harzianum and the compost separately (Spanish Pending Patent of Microgaia Biotech). The inoculation of compost with suppressive microorganisms such as Trichoderma sp. improves the suppressive efficacy of composts, decreasing the disease and enhance the plant health (López-Mondéjar et al., 2010).

ARBUSCULAR MYCORRHIZAL FUNGI (AMF – © Symbio-m) :

Some fungi naturally present in soil form a mutualistic relationship with the roots of the plants, called symbiosis. This partnership was invented by nature itself, to help plants grow better and be more resistant to environmental stress. Thanks to their incredible ability to connect to plant roots, the microscopic fungal fibres vastly extend the root system. They extract water and nutrients from a large volume of surrounding soil, and bring them to the plant, improving nutrition and growth. One of the most important abilities of mycorrhizal fungi is that they stay attached to the roots and support the plant for its entire life. Moreover, plants with mycorriza are often more resistant to diseases, such as those caused by microbial soil-borne pathogens, and are also more resistant to the effects of drought.

The Czech company Symbiom is specialist in the production of mycorrizal products used to supply the plant with much more extra water and nutrients, contributing to superior growth, resistance and health of many crops.

Mycorrhiza form a beneficial soil microbe–plant interaction that affects the physiological traits of many crop plants, including yield and food-quality. Recently, mycorrhiza have become a prospective tool for sustainable, low input crop produc systems. Mycorrhizas inhabit the ecosystems of terrestrial plants as a natural way to acquire nutrients from soils (Pirozynski and Dalpe 1992). The majority of crop plants form relationships with arbuscular mycorrhizal (AM) fungi, and thein responsiveness to mycorrhiza depends on numerous factors including genotype, soil-related and climate properties, and agrochemical inputs into the cultivation system. Extensive mycorrhizal research conducted during the last few decades has focused on the mechanisms of the symbiosis and its role in the management of a sustainable crop production where mycorrhiza could play an important role in combating these problems. (rev. by Vosátka and Albrechtová 2008). Example of already successfully developer technology of organic or semi-organic cultivation of blueberry plants developer by Symbio-m in already finished project EUREKA is given in illustrations bellow.

Mycorrhiza’s problem-solving potential applies to (1) increasing agronomic production, (2) enhancing carbon sequestration in terrestrial ecosystems to stabilize the atmospheric CO2 concentration, (3) converting degraded, polluted or desertified soils to restored land or sustainable agroecosystems, and (4) developing sustainable farming/cropping systems aimed at improving water use efficiency and soil properties to vombat increasing erosion and minimize risks of water pollution and eutrophication. Thus, AM fungi are regarded as essential components of sustainable soil–plant systems (Schreiner et al. 2003). As an ecological biofertilizer, a bioprotectant against environmental stresses, an agent controlling root pathogens and a soil improver acting as an anti-erosion agent, mycorrhiza possesses great potential in sustainable or organic agriculture. Moreover, it appears that its contribution to carbon sequestration is substantial; mycorrhizal fungi can be an important soil

carbon sink and often constitute 20–30% of total soil microbial biomass (Leake et al. 2004). Under conditions of continuously increasing ambient CO2 concentrations, AM fungi are expected to increase their role..

CHANGES IN FOOD QUALITY PROPERTIES OF PLANTS and TOLERANCE AGAINST ROOT PATHOGENS INDUCED BY MYCORRHIZA

One of the main potentials of mycorrhizas for sustainable agriculture is that they induce plant physiological changes that affect the quality and safety of food crops, including a higher production of antioxidants or essential oils, or reduced uptake of pollutants such as heavy metals to plant tissues (Toussaint 2007; Toussaint et al. 2007; Vosa´tka and Albrechtova´ 2008, Geneva et al. 2010). Although no clear mechanism other than an improvement in the nutritional status (mainly P) has been proposed (Toussaint 2007), yet the beneficial fungus–plant interactions has shown enhancement in productivity of crops by synthesizing an increased level of active compounds (Rai et al. 2001). For example, the suitable selection of host plant–fungus genotype led to an altered accumulation of essential oil levels in AM-colonized plants of Mentha arvensis (Freitas et al. 2004) and sweet basil Ocimum basilicum L. (Copetta et al. 2006, 2007; Toussaint et al. 2007). Thus, the use of mycorrhiza is being extended to herbal plants in order to get maximum benefits by the steadily increasing herbal medicine industry (Ernst 2000).

Recent research shows also increase in antioxidant properties of harvested plant parts what increases biological value of mycorrhiza in agricultural and horticulatural production. For exapmle, It was found that the application of microbial consortia (Pseudomonas + Azotobacter + Azosprillum + AMF) treatment had the most effect on lycopene, antioxidant activity and potassium contents on tomato (Ordookhani et al. 2010). Tolerance to Fusarium wilt and anthracnose and the changes in antioxidative abilities in mycorrhizal strawberry plants were observed in strawberry (Fragaria x ananassa Duch., 'Nohime') plants inoculated with AMF (Li et al., 2010). Recently Symbio-m presented results from our ongoing research at conference of ongoing COST project in Evora (see the poster below, Latr et al. 2010).

Arbuscular mycorrhizal fungi (AMF) can act also as bioprotector agents against different pathogens, as illustrated below on experiments conducted in Spain by professor Barea. For example, AMF acts as bioprotector agents against wilt induced by Verticillium spp. in pepper (e.g. Goicoechea et al. 2010,