When do arbuscular mycorrhizal fungi protect plant roots from pathogens?

Arbuscular mycorrhizal (AM) fungi are mainly thought to facilitate phosphorus uptake in plants, but they can also perform several other functions that are equally beneficial. Our recent study sheds light on the factors determining one such function, enhanced plant protection from root pathogens. Root infection by the fungal pathogen Fusarium oxysporum was determined by both plant susceptibility and the ability of an AM fungal partner to suppress the pathogen. The non-susceptible plant species (Allium cepa) had limited F. oxysporum infection even without AM fungi. In contrast, the susceptible plant species (Setaria glauca) was heavily infected and only AM fungi in the family Glomeraceae limited pathogen abundance. Plant susceptibility to pathogens was likely determined by contrasting root architectures between plants, with the simple rooted plant (A. cepa) presenting fewer sites for infection. AM fungal colonization, however, was not limited in the same way in part because plants with fewer, simple roots are more mycorrhizal dependent. Protection only by Glomus species also indicates that whatever the mechanism(s) of this function, it responds to AM fungal families differently. While poor at pathogen protection, AM fungal species in the family Gigasporaceae most benefited the growth of the simple rooted plant species. Our research indicates that plant trait differences, such as root architecture can determine how important each mycorrhizal function is to plant growth but the ability to provide these functions differs among AM fungi.Key words: arbuscular mycorrhizal fungi, Fusarium oxysporum, root architecture, pathogen protection, multi-functionalityArbuscular mycorrhizas (AM) represent the oldest and most widespread symbiosis with land plants.¹ Most mycorrhizal research has focused on the ability of AM fungi to facilitate nutrient uptake, particularly phosphorus.² Although researchers recognize that AM fungi are multi-functional,³ it is not clear what factors determine which function an AM fungus performs or its relative importance to the plant.⁴ Newsham et al. (1995)³ hypothesized that AM function is based on root architecture: plants with simple rooting systems are dependent on mycorrhizas for nutrient uptake, while those with complex root systems are less dependent on mycorrhizas for nutrient uptake, but are more susceptible to root pathogens because of increased numbers of infections sites.³ These two functions, phosphorus uptake and enhanced pathogen protection from mycorrhizas also depend on the identity of the fungus. Arbuscular mycorrhizal fungi in the family Gigasporaceae are more effective at enhancing plant phosphorus, while AM fungi in the Glomeraceae better protect plants from root pathogens.⁵Our results support both plant and fungal control of a common pathogen, Fusarium oxysporum, and the interaction between these two factors ultimately determined the level of pathogen infection and plant mycorrhizal benefit. We inoculated two plant species that have contrasting root architectures with one of six AM fungal species from two families (or no AM fungi). After five months of growth, plants were inoculated with F. oxysporum, grown for another month and then harvested. All plant seeds and fungi were collected in a local old field community.⁶ Allium cepa (garden onion) was not susceptible to F. oxysporum likely because it has only a few adventitious roots below the main bulb that do not present many sites for infection. In contrast, Setaria glauca (yellow foxtail) was heavily infected by F. oxysporum and has fine roots with increased numbers of branching points and lateral meristems where fungi can colonize.⁷ For the susceptible plant (S. glauca), AM fungal species from the family Glomeraceae were effective at reducing pathogen abundance while species from the Gigasporaceae were not. Forming a symbiosis with a Glomus species resulted in S. glauca plants that were as large as control plants. AM fungal species from the family Gigaspoaceae were more beneficial to growth of the simple rooted A. cepa, which had fewer roots to take up soil nutrients.Reduced rooting structures may limit pathogen infection sites, but AM fungal colonization was not limited in the same way and may actually alter plant root architecture. While the simple rooted A. cepa had limited pathogen susceptibility, it had twice the AM fungal colonization of the complex rooted S. glauca. Because the simple rooted plant has a greater dependence on mycorrhizas,⁸ it likely transmits chemical signals to rapidly initiate mycorrhizal formation,⁹ but then may have less control on the spread of AM fungi within the root. In contrast, S. glauca is more susceptible to fungal pathogens and may be less mycorrhizal dependent in nature.¹⁰ As a result, S. glauca may treat all colonizing root fungi as potential parasites. Colonization by AM fungi from the Glomeraceae was also much greater than those in the Gigasporaceae due to differences in fungal life history strategy between these families.^11,12 AM fungal colonization can reduce root branching in plants and alter plant allocation to roots, thereby increasing mycorrhizal dependence for nutrients^10,13 and potentially reducing pathogen infection sites. Mycorrhizal induced changes to plant root architecture may therefore reinforce current mycorrhizal associations and alter future fungal colonization attempts.¹⁴ An important next step is to test if AM fungal families (or species) alter plant root architecture in different ways and the degree to which these effects depend on colonization timing and the plant host.Our study did not isolate the particular mechanism by which AM fungi control pathogens, but this mechanism clearly differentiates between AM fungal families. AM fungi can control pathogens through several mechanisms including direct competition for colonization sites, indirect initiation of plant defensive responses or altering other rhizosphere biota.¹⁵ Although these AM fungal families differ in the intensity of root colonization,¹¹ percentage of root length colonized by an AM fungus is a poor predictor of pathogen limitation compared to family identity,^12,16 suggesting that direct competition for space is unlikely. AM fungi share many cell surface molecules with pathogenic fungi like Fusarium.¹⁷ These molecules can act as signals that initiate plant production of defensive compounds such as phytoalexins, phenolics and other compounds.¹⁸ While AM fungi appear to evade these defenses, only AM fungal species in the family Glomeraceae would have elicited plant responses which altered future infection by F. oxysporum. AM fungi in the Gigasporaceae may differ more from F. oxysporum in their chemical signals or not colonize roots sufficiently to induce a sustained, system-wide plant response. In addition, many rhizosphere related microbes are antagonistic to pathogenic fungi¹⁵ and may differ in their response to the different AM fungal families.¹⁹ Because rhizosphere microbes also differ among plant species, plant pathogen protection may be influenced by multiple ecological interactions that determine the specific cases when mycorrhizal pathogen protection occurs. To distinguish between these mechanisms, future experiments could test whether biochemical similarity or ecological similarity (especially with other soil biota) between an AM fungus and fungal pathogen can predict mycorrhizal induced pathogen protection.Plant and fungal identity clearly affect AM fungal function and benefit, but to accurately use AM fungi in agriculture and restoration^20,21 we must clearly understand how functional mechanisms differ. Different mycorrhizal functions may be based on common plant traits like root architecture, but ecology, colonization timing and environment may alter the specific function AM fungi provide and its importance to plants. While it may be useful to establish greenhouse rules about which fungal species perform specific mycorrhizal functions, predicting their role in more complex systems relies on understanding if other factors will enhance or negate these effects. Most AM fungal species vary in their ability to perform each function and these can be locally adapted to limiting soil nutrients.²² In plants, there is also a range to which specific mycorrhizal functions may benefit plant fitness, and these responses are based on both plant traits (which change throughout a plant''s life cycle) and the local environment.^23,24 Given this variation, it is critical to understand if AM fungi can respond to cues from the plant or the environment to identify what factors limit plant growth and whether a the most effective AM fungus shows a greater response.     

New insights into the signaling pathways controlling defense gene expression in rice roots during the arbuscular mycorrhizal symbiosis

Mycorrhizal fungi form a mutualistic relationship with the roots of most plant species. This association provides the arbuscular mycorrhizal (AM) fungus with sugars while the fungus improves the uptake of water and mineral nutrients in the host plant. Moreover, the induction of defense gene expression in mycorrhizal roots has been described. While salicylic acid (SA)-regulated Pathogenesis-Related (PR) proteins accumulate in rice roots colonized by the AM fungus G. intraradices , the SA content is not significantly altered in the mycorrhizal roots. Sugars, in addition to being a source of carbon for the fungus, might act as signals for the control of defense gene expression. We hypothesize that increased demands for sugars by the fungus might be responsible for the activation of the host defense responses which will then contribute to the stabilization of root colonization by the AM fungus. An excessive root colonization might change a mutualistic association into a parasitic association.Key words: Glomus intraradices, glucose, fructose, Oryza sativa, pathogenesis-related (PR), salicylic acid (SA), sucrose, sugarsThe arbuscular mycorrhizal (AM) fungi are obligate biotrophs that establish mutualistic associations with the roots of over 90% of all plant species. AM fungi improve the uptake of water and mineral nutrients in the host plant, mainly phosphorus and nitrogen, in exchange for sugars generated from photosynthesis. The benefits of the AM symbiosis on plant fitness are largely known, including increased ability to cope with biotic and abiotic stresses.^1,2 In fact, the amount of carbon allocated to mycorrhizal roots might be up 20% of the total photosynthate income.³ During root colonization, the AM fungus penetrates into the root through the epidermal cells and colonizes the cortex. In the root cortical cells, the fungus forms highly branched structures, called arbuscules, which are the site of the major nutrient exchange between the two symbionts.^4,5 The legumes Medicago truncatula and Lotus japonicus have been widely adopted as the reference species for studies of the AM symbiosis. Cereal crops and rice in particular are also able to establish symbiotic associations with AM fungi.^6,7 Arabidopsis thaliana, the model system for functional genomics in plants, has no mycorrhization ability.It is also well known that plants have evolved inducible defense systems to protect themselves from pathogen invasion. Challenge with a pathogen activates a complex variety of defense reactions that includes the rapid generation of reactive oxygen species (ROS), changes in ion fluxes across the plasma membrane, cell wall reinforcement and production of antimicrobial compounds (e.g., phytoalexins).⁸ One of the most frequently observed biochemical events following pathogen infection is the accumulation of pathogenesis-related (PR) proteins.⁹ For some PR proteins antimicrobial activities have been described (e.g., chitinases, β-1,3-glucanases, thionins or defensins). The plant responses to pathogen attack are activated both locally and systemically. The phytohormones salicyclic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) act as defense signaling molecules for the activation of defense responses.¹⁰ Whereas SA-dependent signaling often provides resistance to biotrophic pathogens, JA/ET-dependent signaling is effective against necrotrophic pathogens.¹¹ During plant-pathogen interactions, cross-talk between SA and JA/ET signaling pathways provides the plant with the opportunity to prioritize one pathway over another to efficiently fine-tune its defense response to the invading pathogen. Contrary to biotrophic pathogens which exhibit a high degree of host specificity, the AM fungi manage to colonize a broad range of plant species.Evidence also exists on the existence of common mechanisms and signaling pathways governing responses to AM and pathogenic fungi.^2,12,13 Alterations in the content of hormones acting as defense signals also appear to occur during the AM symbiosis. As an example, JA and its derivatives (jasmonates) are believed to play an important role during the AM symbiosis in M. truncatula or tomato plants.^14,15 However, controversial data exists in the literature concerning the involvement of the various defense-related hormones during AM functioning. In particular, our current understanding of SA signaling during AM symbiosis is not clear.We recently documented the symbiotic proteome of the rice roots during their interaction with the AM fungus Glomus intraradices.⁶ A majority of the proteins identified in the rice symbiotic proteome are proteins with a function in defense responses or sugar metabolism. Among the proteins that accumulated at high levels in mycorrhizal rice roots compared to non mycorrhizal roots were PR proteins belonging to different PR families, such as PR1, chitinases (PR3), PR5 and several PR10 proteins. The PR1 and PBZ1 (a member of the PR10 family of PR proteins) genes are considered markers of the activation of defense responses in rice plants.^16,17 Of interest, the expression of many of the AM-regulated PR genes was previously reported to be induced by SA.^16,18–20 Proteins acting as oxidative stress protectors, such as ascorbate peroxidases, peroxidases and glutathione-S-transferases, also accumulated in mycorrhizal rice roots. Together, these observations support that the plant''s immune system is activated in the mycorrhizal rice root.To gain further insights into the molecular mechanisms governing PR gene expression in mycorrhizal roots, the SA and sugar contents of mycorrhizal roots were determined. Towards this end, rice (Oryza sativa ssp. japonica cv. Senia) plants were inoculated with the AM fungus G. intraradices.⁶ At 42 days post-inoculation (dpi), the overall colonization of the rice roots ranged from 25 to 30% as judged by microscopical observations of trypan blue-stained roots (results not shown; similar results were reported previously in reference ⁶). By this time, all the events related to fungal development, namely intraradical hyphae, arbuscules at different morphological stages of formation and vesicles, were present in G. intraradices-inoculated roots, thus confirming the establishment of the symbiotic association in the rice roots.Knowing that many AM-regulated proteins are also regulated by SA in rice roots, it was of interest to determine whether the level of endogenous SA increases in mycorrhizal roots compared to non mycorrhizal roots. In plants, intracellular SA is found predominantly as free SA and its sugar conjugate SA-glucoside (SAG). Root samples were analyzed for SA content, by measuring the level of both free SA and SAG as previously described in reference ²¹. This analysis revealed no significant differences, neither in free nor in SAG, between mycorrhizal and non mycorrhizal roots (Fig. 1). Then, it appears that although the expression of PR genes (functioning in a SA-dependent manner) is activated during the AM symbiosis, the fungus G. intraradices do not exploit the SA-mediated signaling pathway for induction of PR genes.Open in a separate windowFigure 1SA content, free SA and SA-glucoside (SAG) conjugate, in roots of mock-inoculated (−Gi) and G. intraradices-inoculated (+Gi) rice plants. SA determination was carried out at 42 days post-inoculation with G. intraradices. Three independent biological samples and three replicates per biological sample were used for quantification of SA. Two out of the three samples were the same ones used for the characterization of the symbiotic proteome in which the accumulation of SA-regulated PR genes was observed in reference ⁶. FW, fresh weight. Bars represent the means ± standard error.On the other hand, a direct link between sugar metabolism and the plant defense response has been established, including the phenomenon of high sugarmediated resistance and the finding that various key PR genes are induced by sugars. Transgenic approaches that lead to alterations in photoassimilate partitioning, either sucrose or hexoses, also alter PR gene expression.^22,23 In other studies, a SA-independent induction of PR genes by soluble sugars, sucrose, glucose and fructose, was reported in reference ²⁴.Sucrose, the main form of assimilated carbon during photosynthesis, is transported to the root tissues via the phloem where it becomes available to the root cells. As previously mentioned, characterization of the rice symbiotic proteome revealed alterations in the accumulation of proteins involved in sugar metabolism, such as enzymes involved in glucolysis/gluconeogenesis (e.g., fructose-1,6-bisphophate aldolase, enolase) or in pentose interconversions (e.g., UDP-glucose dehydrogenase).⁶ Because the plant provides sugars to the fungus, it is not surprising to find alterations in enzymes involved in sugar metabolism in the mycorrhizal roots. Evidence also supports that AM fungi acquire hexoses from the host cell and transform it into trehalose and glycogen, the typical sugars in the fungus.²⁵ Utilization of sucrose then requires hydrolysis in the plant cell which can be performed by sucrose synthase, producing UDP-glucose and fructose or invertases, producing glucose and fructose. Along with this, increased activities of invertases and sucrose synthases or increased expression of their corresponding genes, have been described during AM symbiotic interactions.^26,27 Very recently, the MtSucS1 sucrose synthase gene was reported to be essential for the establishment and maintenance of the AM symbiosis in Medicago truncatula.²⁸ In this context, we decided to explore whether colonization by G. intraradices has an effect on the accumulation of soluble sugars in rice roots.Sucrose, glucose and fructose content were measured enzymatically²³ in the rice roots at 42 days post-inoculation with G. intraradices . A tendency to a higher sucrose level was observed in mycorrhizal roots compared to non-mycorrhizal roots (Fig. 2). Concerning the hexose content, the mycorrhizal roots had a significantly lower hexose, both glucose and fructose levels, compared to non-mycorrhizal roots (p ≤ 0.05, Fig. 2). This finding is in agreement with results reported by other authors indicating that the fungal symbiont takes up and uses hexoses within the root.^29,30 The observation that the sucrose content is not significantly affected by mycorrhiza functioning, indicates that the host cell is able to sense sucrose concentration in order to maintain it at sufficient but constant levels to satisfy the demand for sugars by the fungal symbiont.Open in a separate windowFigure 2Sugar content in roots of rice plants inoculated with G. intraradices (+Gi) or mock-inoculated (−Gi). (A) Sucrose content. (B) Glucose content. (C) Fructose content. Measurements were made at 42 days post-inoculation with G. intraradices. Bars represent the means ± standard error.Clearly, the outcome of the AM symbiosis is an overall improvement of the fitness of both partners: the plant supplies the fungus with photosynthates whereas the fungus delivers nutrients from the soil to the host plant. Variations in the extent of colonization of the AM fungi will impose different carbon demands on the plants. However, a high demand of photosynthates by the mycorrhizal root might result in increased mycorrhization which, in turn, might be detrimental for the host plant. The rate of colonization and the amount of fungal biomass must then be tightly controlled by the host plant. We postulate that an increased sink strength by AM colonization might result in transient and/or localized increases in sugar concentrations in the root cell which might be the signal for the activation of defense gene expression. A schematic representation of plant responses associated with increased demands for sugars and deployment of defense responses is shown in Figure 3. According to this model, sugars might play a dual role during the AM symbiosis: (1) sugars are transferred from the plant to the fungus in exchange of mineral nutrients and (2) sugars alter host gene expression, leading to the activation of defense-related genes. This will allow the host plant to avoid an excessive root colonization by the AM fungus that might cause negative effects on the plant''s fitness. A complex exchange and interplay of signals between plant roots and AM fungi must then operate during functioning of the AM symbiosis for coordination of joint nutrient resource explotation strategies and control of the plant''s immune system. During evolution, co-adaptation between the two symbionts, the AM fungi and the host plant, must have occurred for stabilization of mycorrhizal cooperation and optimal functioning of mycorrhizal associations along the mutualism-parasitism continuum.Open in a separate windowFigure 3Proposed model for a sugar mediated-activation of defense-related genes in mycorrhizal roots. In the arbuscular mycorrhizal symbiosis, the fungal symbiont colonizes root cortical cells, where it establishes differentiated hyphae called arbuscules. Arbuscules are the site of mineral nutrient transfer to the plant and the site of carbon acquisition by the fungus. Although arbuscules form within the root cortical cells, they remain separated from the plant cell cytoplasm by a plant-derived membrane, the periarbuscular membrane. In this way, an interface is created between the plant and fungal cells which appears to be optimal for nutrient transfer. Sucrose is transported through the phloem into the root. In the root cell, sucrose is hydrolyzed by host invertase and sucrose synthase activities before uptake by the AM fungus. Hexose uptake at the plant-fungus interfase might be passive with a concentration gradient maintained by rapid conversion of hexoses taken up by the fungus to trehalose and glycogen. Active mechanisms might also operate for hexose transport processes between the host cell and the symbiont. Under conditions of a high demand for sugars by the AM fungus, transient increases in sugar content will occur in the root cells which would be the signal for the activation of the host defense responses. The host-produced defense compounds would stabilize the level of root colonization by the AM fungus. An excessive root colonization might change the mutualistic association into a parasitic one.     

Piriformospora indica mycorrhization increases grain yield by accelerating early development of barley plants

Root colonization by the basidiomycete fungus Piriformospora indica induces host plant tolerance against abiotic and biotic stress, and enhances growth and yield. As P. indica has a broad host range, it has been established as a model system to study beneficial plant-microbe interactions. Moreover, its properties led to the assumption that P. indica shows potential for application in crop plant production. Therefore, possible mechanisms of P. indica improving host plant yield were tested in outdoor experiments: Induction of higher grain yield in barley was independent of elevated pathogen levels and independent of different phosphate fertilization levels. In contrast to the arbuscular mycorrhiza fungus Glomus mosseae total phosphate contents of host plant roots and shoots were not significantly affected by P. indica. Analysis of plant development and yield parameters indicated that positive effects of P. indica on grain yield are due to accelerated growth of barley plants early in development.Key words: mycorrhiza, barley development, Piriformospora indica, phosphate uptake, grain yield, pathogen resistanceThe wide majority of plant roots in natural ecosystems is associated with fungi, which very often play an important role for the host plants'' fitness.¹ The widespread arbuscular mycorrhizal (AM) symbiosis formed by fungi of the phylum Glomeromycota is mainly characterized by providing phosphate to their host plant in exchange for carbohydrates.^2,3 Fungi of the order Sebacinales also form beneficial interactions with plant roots and Piriformospora indica is the best-studied example of this group.⁴ This endophyte was originally identified in the rhizosphere of shrubs in the Indian Thar desert,⁵ but it turned out that the fungus colonizes roots of a very broad range of mono- and dicotyledonous plants,⁶ including major crop plants.^7–9 Like other mutualistic endophytes, P. indica colonizes roots in an asymptomatic manner¹⁰ and promotes growth in several tested plant species.^6,11,12 The root endophyte, moreover, enhances yield in barley and tomato and increases in both plants resistance against biotic stresses,^7,9 suggesting that application in agri- and horticulture could be successful.     

Assessing fungal root colonization for plant improvement

Fungal endophytes display a broad range of symbiotic interactions with their host plants. Current studies on their biology, diversity and benefits are unravelling their high relevance on plant adaptation to environmental stresses. Implementation of such properties may open new perspectives in agriculture and forestry. We aim to exploit the endophytic capacities of the fungal species Fusarium equiseti, a naturally occurring root endophyte which has shown antagonism to plant pathogens, and Pochonia chlamydosporia, a nematophagous fungus with putative endophytic behavior, for plant protection and adaptation to biotic and abiotic stress. A real-time PCR protocol for quantification of the fungal population, together with Agrobacterium-mediated genetic transformation with the GFP gene for confocal microscopy analyses, were designed and applied to assess endophytic development of both these fungal species. Although quantification of both F. equiseti and P. chlamydosporia yielded similar degrees of root colonization, microscopical observations demonstrated differences in infection and development patterns. Furthermore, we found evidences of plant response against endophyte colonization, supporting a balanced antagonism between the endophyte virulence and the plant defenses. Optimization and application of the methodologies presented herein will allow elucidation of beneficial interactions among these endophytes and their host plants.Key words: endophytes, Fusarium equiseti, fungal detection, mutualism, Pochonia chlamydosporia, root colonizationTissues of nearly all plants in natural ecosystems appear to be colonized by endophytic fungi, whose importance in plant development and distribution seem to be crucial, but not yet clearly understood. Fungal endophytes may assist their host plants for adaptation to habitat, protection against biotic or abiotic stresses, plant growth promotion or soil nutrients uptake.^1–5 Recently, biology and function of fungal endophytes in nature has been extensively reviewed,⁶ and these have been grouped into four classes according to their differential interactions with host plants, which may range from mere neutralism to an active mutualism.Exploitation of beneficial properties of endophytes is of great relevance at an applied level, either to increase production yields of agricultural crops, control of plant diseases or pests, adapt plants to unsuitable growth conditions, or in reforestation activities. In this sense, we aim to exploit the endophytic capacities of selected fungi from two independent approaches: through the use of natural endophytes, which have shown to confer benefits to their host plants,⁵ or via a putative endophytic colonization of plant tissues by fungi with desirable properties. The latter strategy has been already introduced for several biological control agents such as nematophagous fungi colonizing roots,⁷ or entomopathogenic fungi in above-ground organs.^8,9 As representatives of each category, we selected two fungal species according to their endophytic capacities and antagonism to root pathogens.^10,11 First, Fusarium equiseti represents naturally occurring endophytes from natural vegetation growing under saline stress conditions.¹² The second, Pochonia chlamydosporia, is a nematode egg-parasite which behaves as a putative endophyte of roots.¹³ Both species have promising properties for application as biocontrol agents of either fungal and/or nematode plant parasites. The achievement of these objectives will depend on an exhaustive knowledge on the endophytic behavior and interactions with the host and other existing microbes, which starts with the assessment of the establishment of fungal populations within plant organs (e.g., roots). In many instances this is a complicated task. Fungal growth is characterized by an implicit irregular development due to the filamentous nature of mycelia, whose complexity increases in structured substrates, such as the interior of plant tissues.These difficulties are enhanced due to uneven distribution of nuclei (from zero to several thousands per cell), physiological activity or vitality of hyphae, and influence of competition among species.¹⁴ As a consequence of this, methods applied traditionally to estimate fungal occurrence within plant tissues (e.g., direct visualization, plating on culture media or immunolocalization^4,15,16) are biased or rather laborious and time consuming. Although applications of molecular methods have been directed to settle these problems, these may also present other weaknesses. Combination of quantitative and qualitative data achieved by both molecular and microscopy methods is probably the best choice to monitor fungi in plant roots, since the advantages of each technique may complement the drawbacks of the other.^14,17We recently optimized and applied both real-time PCR and microscopy techniques to exhaustively study the endophytic development of F. equiseti and P. chlamydosporia in barley roots.¹⁸ The first approach consisted in a specific quantification of nucleic acids from either fungus in roots using Molecular beacon probes.¹⁹ This allows an accurate monitoring of the respective amplicon generation over PCR cyclings, which can be correlated with the amount of fungal biomass.²⁰ Fungal populations detected in roots were statistically similar for both fungal species studied, with an overall colonization of roots which ranged from ca. 5 to 11 ng of fungal target DNA per 100 ng of total DNA. The endophytic proportion of these populations (assessed by surface sterilization of roots prior to DNA extraction) was represented by values between ca. 0.5 to 1 ng of fungal target DNA per 100 ng of total DNA.These quantitative data resulted in an increased sensitivity and dynamic range as compared with traditional culturing methods. Nevertheless, though quantification yielded good results under a gnotobiotic system, where only those fungi of interest are present within plant tissues, implementation for non-axenic semi-field or field experimentation should include modifications. These should cover corrections for presence of other colonizing microorganisms under non-axenic growth conditions-which may contribute to the amount of total DNA extracted from roots-, or variability on DNA extraction yields among samples.²¹ Inclusion of internal standards such as the simultaneous detection of fungal and host plant in a single reaction tube²² would settle these interferences. Multiplex PCR amplification of respective plant or fungal loci would permit detection of differential fluorescent signals emitted by Molecular beacons specific for either target DNA. We are currently optimizing primers and probes designed in Maciá-Vicente et al.¹⁸ for multiplexing with primers and Molecular beacons specific for the host (barley) ubiquitin gene (Fig. 1).Open in a separate windowFigure 1Amplification by conventional PCR of DNA from plant (h; barley), endophyte (e; P. chlamydosporia), or endophyte-colonized plants (h + e). For this, single locus detection with primers specific for the fungal alkaline serine protease p1,¹⁸ (Fp) and the plant ubiquitin (Pp) is shown, and multiplex PCR using both primer pairs (Fp + Pp) for one-tube simultaneous detection.In addition to real-time PCR assays, endophytic behavior of F. equiseti and P. chlamydosporia was assessed using live-cell microscopy. Aiming to develop techniques for further studies in semi-field experiments, we generated genetically transformed isolates for both F. equiseti and P. chlamydosporia, with constitutive expression of the fluorescent reporter protein GFP. For this purpose an Agrobacterium tumefaciens-mediated protocol was applied due to its efficiency in fungal transformation.²³ Observation of barley roots inoculated with GFP-tagged isolates under laser scanning confocal microscopy permitted a time-course qualitative monitoring of the infection processes by F. equiseti and P. chlamydosporia, which displayed different endophytic patterns. Loading studies with the endocytotic tracker FM4-64 allowed a discrimination of new traits of fungal colonization of roots. Fungal hyphae appeared tightly fitted in a plant membrane-derived sheath during the first invasive stages, in a similar manner to that which occurs during pathogenesis,²⁴ but also in mutualistic interactions,²⁵ indicating it may be (at least originarly) an unspecific barrier to fungal invasion. However, this membrane was lost with time, as a consequence of hyphal aggressiveness over plant cell infection.Differential wavelength emission between GFP and FM4-64 also allowed detection of a heterogeneous distribution of viable and non-viable hyphae within the root cortex, the latter linked to plant defense responses such as abundant production of papillae or vacuoles. This colonization pattern suggests that endophytic interaction established by both fungi is a “push and pull” balance between hyphal growth and the capacity of the plant to get rid of the invader. Yet we do not know whether remaining undegraded nucleic acids contents of within-cortex dead hyphae may contribute to fungal target DNA detection by real-time PCR. Although this fact may represent a bias for endophytes quantification, combination with microscopical analyses complements the information achieved.We are currently investigating practical and theoretical aspects of the root inoculation of fungi with endophytic capabilities and antagonistic potential to root pathogens (fungi and nematodes). This research also includes evaluating root colonization abilities for different host plants, both monocots and eudicots. As an example, root colonization efficiency by P. chlamydosporia dramatically changes between barley and tomato. In the latter, hyphae within roots are sparse and restricted to epidermal root cells,¹³ with no evident connection between infected cells (Fig. 2A). In spite of this restricted distribution, fungal chlamydospores (the propagules usually found in P. chlamydosporia-harboring soils) may be frequently found on the root surface. Their viability (according to GFP expression in the cytoplasm), irrespective of that of surrounding hyphae (Fig. 2B), could support that root colonization creates a stable source of inoculum to sustain the populations of the microorganism in the rhizospheric soil. Our final goal is to optimize biocontrol performance and crop growth promotion by endophytes under agricultural conditions.Open in a separate windowFigure 2Laser scanning confocal microscopy images of one-month-old tomato roots colonized by GFP-tagged P. chlamydosporia. (A) P. chlamydosporia hypha restricted to a single epidermal root cell. (B) Chlamydospore in the root surface. Note GFP fluorescence within cells (viable) in contrast to lack of fluorescence in peduncle (non-viable). Bars = 20 µm.     

Calcium opens the dialogue between plants and arbuscular mycorrhizal fungi

Calcium ion is considered a ubiquitous second messenger in all eukaryotic cells. Analysis of intracellular Ca²⁺ concentration dynamics has demonstrated its signalling role in plant cells in response to a wide array of environmental cues. The implication of Ca²⁺ in the early steps of the arbuscular mycorrhizal symbiosis has been frequently claimed, mainly by analogy with what firmly demonstrated in the rhizobium-legume symbiosis. We recently documented transient Ca²⁺ changes in plant cells challenged with diffusible molecules released by arbuscular mycorrhizal fungi. Ca²⁺ measurements by the recombinant aequorin method provided new insights into the molecular communications between plants and these beneficial fungi.Key words: legume symbioses, arbuscular mycorrhiza, calcium signalling, fungal signal, plant cell cultures, aequorinIn the rhizosphere plants meet a wide array of microorganisms. In favorable interactions, such as arbuscular mycorrhizal (AM) and nitrogen fixing symbioses, a dialogue is progressively established between the two interacting organisms to make the appropriate partner choice. These two-way communications rely on the interchange of signals released by both potential symbionts. After perception of the signalling molecules, a signal transduction pathway is induced, leading to the activation of the proper genetic and developmental program in both partners.Variations in intracellular free Ca²⁺ concentration occur as one of the initial steps in signalling pathways activated in plants when they encounter pathogens,¹ fungal biocontrol agents² and nitrogen-fixing bacteria.³ Molecules secreted by microorganisms, after binding to specific receptors, trigger in plant cells transient changes in cytosolic Ca²⁺ level, due to the influx of the ion from the extracellular environment and/or the release from internal Ca²⁺ storage compartments.^4,5 Ca²⁺ messages delivered to plant cells are at least partly deciphered on the basis of their spatial and temporal features. The occurrence of different Ca²⁺ signatures guarantees the specificity of the ensuing physiological responses.In the legume-rhizobium symbiosis a definite pattern of Ca²⁺ oscillations has been reported to occur in response to the rhizobial signalling molecule, the Nod factor, in the nucleus and perinuclear cytoplasm of the root hair.⁶ The Ca²⁺ spike number has been recently demonstrated to regulate nodulation gene expression.⁷Legumes are able to engage in a dual symbiotic interaction, with rhizobia and AM fungi. Components of the Ca²⁺-mediated signalling pathway are shared by the two symbioses.⁸ In the mycorrhizal signal transduction pathway the involvement of Ca²⁺ has long been speculated, based on the observed similarities with symbiotic nitrogen fixation.³To evaluate the possible participation of Ca²⁺ in the early steps of the AM symbiosis, we have used a simplified experimental system given by plant cell suspension cultures stably expressing the bioluminescent Ca²⁺-sensitive reporter aequorin.⁹ The use of cultured cells circumvents the problem posed by multilayered organs: in aequorin-transformed seedlings, possible Ca²⁺ changes occurring in rhizodermal cells—the first place where the AM fungal signals are perceived and transduced—can be misrecorded due to luminescence calibration over all root cell layers, resulting in an underestimation of the Ca²⁺ signal in the responsive cells. An experimental design based on challenging host plant cells with the culture medium of different AM fungi (Gigaspora margarita, Glomus mosseae and intraradices) provided the first firm evidence that Ca²⁺ is involved as intracellular messenger during mycorrhizal signalling, at least in a pre-contact stage. Cytosolic Ca²⁺ changes, characterized by specific kinetic parameters, were triggered by diffusates obtained from AM resting and germinating spores,⁹ and extraradical mycelium.¹⁰ Cultured plant cells demonstrated to be competent to perceive the diffusible signal released by AM fungi and to decode the message in a Ca²⁺-dependent pathway. Based on these experiments, it seems that AM fungi announce their presence to the plant through the constitutive release of a chemical signal, even before experiencing the proximity of the plant or its AM symbiotic signals. The notion that the secreted fungal molecules herald, through Ca²⁺, a beneficial message which can be acknowledged only by competent receivers, is supported by: (1) the lack of defense response induction and the upregulation of some genes essential for the AM symbiosis initiation in host plant cells; (2) the unresponsiveness of cultured cells from the nonhost plant Arabidopsis thaliana.Ca²⁺-mediated perception of both AM fungal and rhizobial signals by plant cells unifies the signalling pathways activated in the two symbioses. However, the actual occurrence of Ca²⁺ spiking in AM symbiosis remains to be ascertained, due to limitations of the recombinant aequorin method, when applied to an asynchronous cell population. Contribution of internal Ca²⁺ stores, in particular the nucleus, to the observed Ca²⁺ changes will be a future research goal to be achieved through a pharmacological approach and/or targeting of Ca²⁺ indicators to intracellular compartments.The identification of the plant-derived mycorrhizal signal as strigolactones¹¹ and their inducing activity on AM fungi¹² have represented a major breakthrough in the AM symbiosis research field. Elucidation of the chemical nature of the AM fungal factor, which plays several effects on host plants,^9,13–15 is eagerly awaited.Understanding how AM fungi and rhizobia select compatible plant hosts, thus activating the appropriate symbiotic program, is another facet to be considered in the future to get a complete overview of early signaling events in legume symbioses. Analysis of Ca²⁺ signalling implication in the microbial partner would require the delivery of reliable and sensitive Ca²⁺ probes (such as aequorinor GFP-based¹⁶) for Ca²⁺ measurements in living microorganisms. The recombinant aequorin method has been successfully applied to monitor dynamic changes in intracellular Ca²⁺ levels in the bacteria Anabaena sp.,¹⁷ E. coli,¹⁸ and recently by us in rhizobial strains.¹⁹ Unfortunately, AM fungi have proved not to be amenable to stable transformation, being coenocytic, multinucleate and heterokaryotic,^20,21 and only transient transformants have been obtained so far.^22,23 Further development of the transformation technologies may provide in the future a valuable tool to analyse, from the fungal side, signal perception and transduction during arbuscular mycorrhiza establishment.     

Phytohormones in plant root-Piriformospora indica mutualism

Piriformospora indica is a mutualistic root-colonising basidiomycete that tranfers various benefits to colonized host plants including growth promotion, yield increases as well as abiotic and biotic stress tolerance. The fungus is characterized by a broad host spectrum encompassing various monocots and dicots.^1,2 Our recent microarray-based studies indicate a general plant defense suppression by P. indica and significant changes in the GA biosynthesis pathway.³ Furthermore, barley plants impaired in GA synthesis and perception showed a significant reduction in mutualistic colonization, which was associated with an elevated expression of defense-related genes. Here, we discuss the importance of plant hormones for compatibility in plant root-P. indica associations. Our data might provide a first explanation for the colonization success of the fungus in a wide range of higher plants.Key words: compatibility, plant defense, gibberellic acid, symbiosis, plant hormones     

Lateral root stimulation in the early interaction between Arabidopsis thaliana and the ectomycorrhizal fungus Laccaria bicolor: Is fungal auxin the trigger?

Lateral root (LR) stimulation during early signal exchange between plant roots and ectomycorrhizal (ECM) fungi has recently been shown to be achieved by modulation of auxin gradients. We suggested that this modulation could occur through altered polar auxin transport (PAT) and through activation of auxin signalling pathways in the root. However, it remains unclear, which fungal molecules alter auxin pathways inside the plant partner. It has been suggested in previous studies that auxin released by the fungus could trigger observed plant responses during early signal exchange and later on during root colonization. Here we focus on the early interaction and we provide evidence for an alternative mechanism. Indeed, LR stimulation by the fungus in Arabidopsis thaliana followed a totally different timing than with exogenously applied auxin. Furthermore, experimental conditions that excluded the exchange of soluble molecules while allowing exchange of volatile(s) between the plant and the fungus were sufficient for LR induction, therefore questioning the role of secreted fungal auxin. These data suggest that volatiles released by the fungus and sensed by the plant may act upstream of altered auxin signaling in the plant.Key words: mycorrhiza, ectomycorrhiza, lateral root, auxin, volatiles, ethylene, jasmonic acidInteractions of plant roots with symbiotic, ectomycorrhizal soil fungi lead to lateral root (LR) stimulation during the very early interaction phase.¹ This LR stimulation has recently been shown to be independent of root colonization and to occur as well in non-mycorrhizal plants, such as Arabidopsis suggesting that fungal signals have a broad perception spectrum.^1,2 However, little is known about the type of signals exchanged between fungi and their plant partners during this early interaction phase. Several studies have proposed a role for the phytohormone auxin produced and secreted by ECM fungi as the signalling molecule during ECM fungus/plant signaling.^2–7 Recently we studied changes in auxin response and auxin transport in poplar and Arabidopsis thaliana roots during contact with the ECM fungus Laccaria bicolor.¹ We demonstrated that the presence of the fungus enhances the auxin response and distribution at the root apex and that this, as well as LR stimulation, is reliant on polar auxin transport through AtPIN2 and probably through PtPIN9 in poplar. Here, using Arabidopsis thaliana, whose LR stimulation by Laccaria bicolor has been demonstrated, we propose that not yet identified fungal volatiles may regulate auxin homeostasis in the plant, questioning the contribution of the auxin released by the fungus on the induction of LR.     

pH signature for the responses of arbuscular mycorrhizal fungi to external stimuli

Environmental and developmental signals can elicit differential activation of membrane proton (H⁺) fluxes as one of the primary responses of plant and fungal cells. In recent work,¹ we could determine that during the presymbiotic growth of arbuscular mycorrhizal (AM) fungi specific domains of H⁺ flux are activated by clover root factors, namely host root exudates or whole root system. Consequently, activation on hyphal growth and branching were observed and the role of plasma membrane H⁺-ATPase was investigated. The specific inhibitors differentially abolished most of hyphal H⁺ effluxes and fungal growth. As this enzyme can act in signal transduction pathways, we believe that spatial and temporal oscillations of the hyphal H⁺ fluxes could represent a pH signature for both early events of the AM symbiosis and fungal ontogeny.Key words: H⁺-specific vibrating probe, pH signatures, arbuscular mycorrhiza, pH signalling, Gigaspora margaritaThe 450-million-year-old symbiosis between the majority of land plants and arbuscular mycorrhizal (AM) fungi is one of the most ancient, abundant and ecologically important symbiosis on Earth.^2,3The development of AM interaction starts before the physical contact between the host plant roots and the AM fungus. The hyphal growth and branching are induced by the root factors exudated by host plants, followed by the formation of appressorium leading to the hyphal penetration in the root system. These root factors seems to be specifically synthesized by host plants, since exudates from non-host plants are not able to promote neither hyphal differentiation nor appressorium formation.^4,5 Most root exudates contain several host signals or better, active compounds including flavonoids^6,19 and strigolactones,^7,8 however many of them are not yet known.Protons (H⁺) may have an important role on the fungal growth and host signal perception.¹ In plant and fungal cells, H⁺ can be pumped out through two different mechanisms: (1) the activity of the P-type plasma membrane (PM) H⁺-ATPase⁹ and (2) PM redox reactions.¹⁰ The proportional contribution from both mechanisms is not known, but in most plant cells the PM H⁺-ATPase seems to be the major responsible by the H⁺ efflux across plasma membrane. AM Fungal cells also energize their PM using P-type H⁺-pumps quite similar to the plant ones. Indeed, some genes codifying isoforms of P-type H⁺-ATPase have been isolated of AM fungi,^11–13 and AM fungal ATP hydrolysis activity was shown by cytochemistry, localized mainly in the first 70 µm from the germ tube tip.¹⁴ This structural evidence correlates with data obtained by H⁺-specific vibrating probe (Fig. 1A and B), which indicates that the H⁺ efflux in Gigaspora margarita is more intense in the subapical region of the lateral hyphae¹ (Fig. 1A). Furthermore, the correlation between the cytosolic pH profile previously obtained by Jolicoeur et al.,¹⁵ with the H⁺ efflux pattern (erythrosine-dependent), seems to clearly indicate that an active PM H⁺-ATPase takes place at the subapical hyphal region. Using orthovanadate, we could show that those H⁺ effluxes are susceptible mainly in the subapical region, but no effect in the apical was found.¹ Recently, a method to use fluorescent marker expression in an AM fungus driven by arbuscular mycorrhizal promoters was published.³¹ It could be adjusted as an alternative to measure “in vivo” PM H⁺-ATPase expression in AM fungal hyphae and their responses to root factors.³¹Open in a separate windowFigure 1(A) H⁺ flux profile along growing secondary hyphae of G. margarita in the presence (open squares) or absence (closed squares) of erythrosin B and its correlation with cytosolic pH (pHc) data described by Jolicoeur et al.,¹⁵ (dotted line). Dotted area depicts the region with higher susceptibility to erythrosin B. (B) ion-selective electrode near to AM fungal hyphae. (C) Stimulation on hyphal H⁺ efflux after incubation with root factors or whole root system. R, roots; RE, root exudates; CO₂, carbon dioxide; CWP, cell wall proteins; GR24, synthetic strigolactone. The medium pH in all treatment was monitored and remained about 5.7, including with prior CO₂ incubation. Means followed by the same letter are statistically equal by Duncan''s test at p < 5%.The H⁺ electrochemical gradient generated by PM H⁺-ATPases provides not only driving force for nutrient uptake,^9,16 but also can act as an intermediate in signal transduction pathways.¹⁸ The participation of these H⁺ pumps in cell polarity and tip growth of plant cells was recently reported,²⁷ addressing their crucial role on apical growth.²⁸ Naturally, in the absence of root factors the AM fungi have basal metabolic^8,21–23 and respiratory activity.²⁴ However when root signals are recognized and processed by AM fungal cells they might become activated.²² We thus searched for pH signatures that could reflect the alterations on fungal metabolism in response to external stimuli. In fact, preliminary analyses from our group demonstrate that AM fungal hyphae increase their H⁺ efflux in response not only to root exudates recognition, but also to other root factors (Fig. 1C). The incubation for 30 min of AM fungal hyphae with several root factors induces hyphal H⁺ efflux similar to the response to intact root system (5 days of incubation). The major increases were found with 1% CO₂ (750%) followed by root cell wall proteins (221%), root exudates (130%) and synthetic strigolactone (5%) (Fig. 1C). Those stimulations could define the transition from the state without root signals to the presymbiotic developmental stage (Fig. 1C). In the case of CO₂, the incorporation of additional carbon could represent a new source of energy, since CO₂ dark fixation takes place in Glomus intraradices germ tubes.^22,25Interestingly, after the treatment with synthetic strigolactone (10⁻⁵ M GR24), no significant stimulation was found compared to the remaining factors (Fig. 1C). It opens the question if the real effect of strigolactone is restrict to hyphal branching and does not intervene in very fast response pathways. Likewise, strigolactones need additional time to exhibit an effect, as recently discussed by Steinkellner et al.,²⁶ However, at the moment, no comprehensive electrophysiological analyses are presently available separating the effects of strigolactone and some flavonoids in AM fungal hyphae.The next target of our work is the study of ionic responses of single germ tubes or primary hyphae to root factors (Fig. 2). As reported by Ramos et al.,¹ we have been observing that the pattern of ion fluxes at the apical zone of primary hyphae is differentiated from secondary or lateral hyphae. In the primary, two interesting responses were detected in the absence of root factors: (1) a “dormant Ca²⁺ flux” and (2) Cl⁻ or anion fluxes at the same direction of H⁺ ions, suggesting a possible presence of H⁺/Cl⁻ symporters at the apex, similarly to what occurs in root hairs (Fig. 2).³⁰ In the presence of root factors such as root exudates the stimulated influxes of Cl⁻ (anion), H⁺, Na⁺ and effluxes of K⁺ and Ca²⁺ are activated. It can explain why the AM fungi hyphal tips are depolarized^20,29 during the period without root signals—“asymbiosis”—as long as K⁺ efflux and H⁺ influx occur simultaneously. Indeed, H⁺ as well as Ca²⁺ ions may act as second messengers, where extra and intracellular transient pH changes are preconditions for a number of processes, including gravity responses and possibly in plant-microbe interactions.^17,30Open in a separate windowFigure 2Ion dynamics in the apex of primary hyphae of arbuscular mycorrhizal fungi. It represents the Stage 1 described in Ramos et al.¹ After treatment with root factors, an activation of Ca²⁺ efflux is observed at the hyphal apex.Clearly, further data on the mechanism of action of signaling molecules such as strigolactones over the signal transduction and ion dynamics in AM fungi will be very important to improve our understanding of the molecular bases of the mycorrhization process. Future studies are necessary in order to provide basic knowledge of the ion signaling mechanisms and their role on the response of very important molecules playing at the early events of AM symbiosis.     

Increased Diversity of Arbuscular Mycorrhizal Fungi in a Long-Term Field Experiment via Application of Organic Amendments to a Semiarid Degraded Soil

In this study, we tested whether communities of arbuscular mycorrhizal (AM) fungi associated with roots of plant species forming vegetative cover as well as some soil parameters (amounts of phosphatase and glomalin-related soil protein, microbial biomass C and N concentrations, amount of P available, and aggregate stability) were affected by different amounts (control, 6.5 kg m⁻², 13.0 kg m⁻², 19.5 kg m⁻², and 26.0 kg m⁻²) of an urban refuse (UR) 19 years after its application to a highly eroded, semiarid soil. The AM fungal small-subunit (SSU) rRNA genes were subjected to PCR, cloning, single-stranded conformation polymorphism analysis, sequencing, and phylogenetic analyses. One hundred sixteen SSU rRNA sequences were analyzed, and nine AM fungal types belonging to Glomus groups A and B were identified: three of them were present in all the plots that had received UR, and six appeared to be specific to certain amendment doses. The community of AM fungi was more diverse after the application of the different amounts of UR. The values of all the soil parameters analyzed increased proportionally with the dose of amendment applied. In conclusion, the application of organic wastes enhanced soil microbial activities and aggregation, and the AM fungal diversity increased, particularly when a moderate dose of UR (13.0 kg m⁻²) was applied.The semiarid Mediterranean areas of Southeastern Spain are affected by environmental degradation and erosive processes due to the fact that they are characterized by a set of climatic conditions that includes irregular and scarce rainfall and long, dry, and hot summers. Under these conditions, the soil organic matter content decreases, and the availability of nutrients and water for plants is reduced. Consequently, soil productivity decreases, levels of below-ground microbially diverse populations decline, and the water deficit limits plant growth so that the vegetation cover of natural soils cannot be sustained. Therefore, the development of revegetation techniques to reduce erosion, to remediate the effects of degradation, and, thus, to allow the restoration of biodiversity is needed. It was previously demonstrated that the application of organic amendments, such as urban refuse (UR), to soil increases the organic matter content of soil and improves the quality and productivity of degraded soils (17, 44, 57). Also, it was previously shown that the organic residues yield an improvement in levels of microbially diverse populations in the soil (43).A substantial part of the soil microbial communities belongs to the arbuscular mycorrhizal (AM) fungi, an ancient group of fungi belonging to the phylum Glomeromycota (49), which form mutualistic associations with the roots of the majority of land plants. These fungi have a variety of beneficial effects on their host plants, such as increasing the uptake of mineral nutrients, particularly phosphorus and nitrogen (41, 52); reduction of pathogen infections (7); improvement of water relations (12) and soil stability (58); and the limitation of heavy metal uptake (34). It is evident that AM fungi are an important factor contributing to the maintenance of terrestrial ecosystem functioning. Studies have shown that the diversity of AM fungal populations in the soil can affect plant diversity and productivity and ecosystem stability (62). Therefore, information on the species composition of the AM fungal community in roots is important for an understanding of mycorrhizal function as well as for the effective management and preservation of the diversity of AM fungal populations in ecological field studies.Thanks to advances in molecular techniques in recent years, it is possible to apply PCR-based molecular methods in order to analyze the diversity of AM fungi colonizing the roots of an individual plant at any given time. Traditional identification based on spore morphology is often problematic, and the abundance of spores in the soil may not accurately reflect AM fungal community composition and dynamics (8). The single-stranded conformation polymorphism (SSCP) approach is a very sensitive and reproducible technique for analyzing the sequence diversity of AM fungi within roots (30). This method is based on nucleotide differences between homologous sequence strands, which are detected by electrophoresis of single-stranded DNA under nondenaturing conditions (38).It is known that the application of organic amendments can have a positive effect on the proliferation of natural AM fungi in crop systems (20, 26). The stimulatory effects of the addition of organic matter on the development of AM fungi could be related to an improvement in the extensive network of AM fungal mycelium in the soil. In this way, the colonized plants are able to effectively exploit nutrients and water from soil (52). Moreover, AM fungi are able to exploit nutrients released by the mineralization of organic matter due to the activities of mineralizing microorganisms (28). However, there are many previous reports that showed a strong negative impact on the presence of AM fungal populations and mycorrhizal colonization when composted urban waste was added to the soil (19, 46). Also, research using trap cultures of host plants showed a decrease in the level of diversity of AM fungal species in soils amended with sewage sludge (25, 61).In a previous study carried out in 1992 at the site that is also the subject of the current work, Roldán and Albaladejo (43) found that the application of UR decreased levels of AM fungal populations in the first year after amendment; however, they observed an increase in levels of these populations 3 years after the addition. We hypothesized that after a long period of time, the application of UR could alter the diversity of AM fungal populations in a highly eroded, semiarid soil and that this effect could be influenced by the refuse application rate. In order to verify this hypothesis, we studied the diversity of the AM fungi associated with the roots of plant species forming the vegetative cover of five plots that received different amounts of UR 19 years after the amendment. Also, we determined whether there was an improvement in soil quality parameters related to soil microbial activity.     

Long-distance transport of signals during symbiosis: Are nodule formation and mycorrhization autoregulated in a similar way?

Legumes enter nodule symbioses with nitrogen-fixing bacteria (rhizobia), whereas most flowering plants establish symbiotic associations with arbuscular mycorrhizal (AM) fungi. Once first steps of symbiosis are initiated, nodule formation and mycorrhization in legumes is negatively controlled by a shoot-derived inhibitor (SDI), a phenomenon termed autoregulation. According to current views, autoregulation of nodulation and mycorrhization in legumes is regulated in a similar way. CLE peptides induced in response to rhizobial nodulation signals (Nod factors) have been proposed to represent the ascending long-distance signals to the shoot. Although not proven yet, these CLE peptides are likely perceived by leucine-rich repeat (LRR) autoregulation receptor kinases in the shoot. Autoregulation of mycorrhization in non-legumes is reminiscent to the phenomenon of “systemic acquired resistance” in plant-pathogen interactions.Key words: arbuscular mycorrhiza, autoregulation, CLE peptides, mutant, nodulation, split-root systemUnder natural conditions, growth of plants is often limited by the availability of nutrients such as nitrogen and phosphorous. Plants have therefore developed strategies to acquire nutrients with the help of soil microorganisms. Most land plants enter mutualistic root symbioses with arbuscular mycorrhizal (AM) fungi, whereas legumes form special root nodules containing nitrogen-fixing bacteria, so-called rhizobia.^1–4 Establishment and maintenance of symbiosis requires plant resources, such as photosynthetically assimilated carbon. To minimize these costs, host plants are able to control the degree of their symbiotic interactions. Above a critical threshold level further establishment of symbiosis is restricted—a feedback phenomenon termed autoregulation of symbiosis. Autoregulation can be experimentally demonstrated in split-root systems. When legume roots are already infected by rhizobia on one side of a split-root, further nodule development is “systemically” inhibited on the other side. Similarly, prior colonization of split-roots by AM fungi on one half suppresses later fungal root colonization on the other half. Hence, important elements of the symbiotic autoregulation circuit are not only localized in roots, but also in aerial parts of the plant, implicating transport of signals in vascular bundles (Fig. 1). Whereas autoregulation of nodulation in legumes has been studied for many decades,^5–9 the first publications clearly stating a shoot-controlled autoregulation of mycorrhization in split-root systems appeared in 2000 for the non-legume barley (Hordeum vulgare) and thereafter for alfalfa (Medicago sativa) and soybean (Glycine max).^10–13 The data from these split-root experiments are supported by the findings that supernodulating (or hypernodulating) loss-of-autoregulation mutants displayed either an increased degree of AM colonization and/or a higher abundance of arbuscules.^14–16Open in a separate windowFigure 1Proposed model of shoot-controlled autoregulation of symbiosis in a split-root system. Prior infection of root A by rhizobia or AM fungi systemically suppresses later establishment of symbiosis in root B. Expression of specific CLE peptides (and/or other peptide hormones) is induced in response to rhizobial nodulation signals (Nod factors) and perhaps also in response to colonization by AM fungi (stage 1). The CLE peptides (and/or other signals) are then presumed to be transported in the xylem to the shoot, where they are perceived by leucine-rich repeat (LRR) autoregulation receptor kinases (stage 2). As a result of autoregulation signaling in the shoot, an unknown shoot-derived inhibitor (SDI) is produced (stage 3) and transported as a phloem-mobile signal to the root. Perception and action of the SDI signal in roots would then inhibit nodulation and root colonization by AM fungi (stage 4).     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

Strigolactones: Internal and external signals in plant symbioses?

As the newest plant hormone, strigolactone research is undergoing an exciting expansion. In less than five years, roles for strigolactones have been defined in shoot branching, secondary growth, root growth and nodulation, to add to the growing understanding of their role in arbuscular mycorrhizae and parasitic weed interactions.¹ Strigolactones are particularly fascinating as signaling molecules as they can act both inside the plant as an endogenous hormone and in the soil as a rhizosphere signal.^2-4 Our recent research has highlighted such a dual role for strigolactones, potentially acting as both an endogenous and exogenous signal for arbuscular mycorrhizal development.⁵ There is also significant interest in examining strigolactones as putative regulators of responses to environmental stimuli, especially the response to nutrient availability, given the strong regulation of strigolactone production by nitrate and phosphate observed in many species.^5,6 In particular, the potential for strigolactones to mediate the ecologically important response of mycorrhizal colonization to phosphate has been widely discussed. However, using a mutant approach we found that strigolactones are not essential for phosphate regulation of mycorrhizal colonization or nodulation.⁵ This is consistent with the relatively mild impairment of phosphate control of seedling root growth observed in Arabidopsis strigolactone mutants.⁷ This contrasts with the major role for strigolactones in phosphate control of shoot branching of rice and Arabidopsis^8,9 and indicates that the integration of strigolactones into our understanding of nutrient response will be complex. New data presented here, along with the recent discovery of phosphate specific CLE peptides,¹⁰ indicates a potential role for PsNARK, a component of the autoregulation of nodulation pathway, in phosphate control of nodulation.     

Piriformospora indica enhances plant growth by transferring phosphate

Piriformospora indica is an endophytic fungus that colonized monocot as well as dicot. P. indica has been termed as plant probiotic because of its plant growth promoting activity and its role in enhancement of the tolerance of the host plants against abiotic and biotic stresses. In our recent study, we have characterized a high affinity phosphate transporter (PiPT) and by using RNAi approach, we have demonstrated the involvement of PiPT in P transfer to the host plant. When knockdown strains of PiPT-P. indica was colonized with the host plant, it resulted in the impaired growth of the host plants. Here we have analyzed and discussed whether the growth promoting activity of P. indica is its intrinsic property or it is dependent on P availability. Our data explain the correlation between the availability of P and growth-promoting activity of P. indica.Key words: Piriformospora indica, phosphate transport, plant growth promotionPhosphorous (P) is one of the most essential mineral nutrients for plant growth and development. In the soil P is present mainly in the form of sparingly soluble complexes that are not directly accessible to plants. Thus, it is the nutrient that limits crop production throughout the world.¹ Plants have therefore evolved a range of strategies to increase the availability of soil P, which include both morphological and biochemical changes at the soil-root interface. For example, increased root growth and branching, proliferation of root hairs, and release of root exudates can increase plant access to inorganic phosphate (Pi) from otherwise poorly available sources.^2,3 Plant root possess two distinct modes of phosphate uptake, direct uptake by its own transporters and indirect uptake through mycorrhizal associations. In plants several high affinity P transporters specifically associated with the uptake of Pi from soil solution. Expression of these transporters is induced in response to P deficiency and enables Pi to be effectively taken up against the large concentration gradient that occurs between the soil solution and internal plant tissues.⁴ However, in arbuscular mycorrhizal associations (indirect uptake), plants acquire Pi from the extensive network of fine extra radical hyphae of fungus, that extend beyond root depletion zones to mine new regions of the soil.⁵ In the case of arbuscular mycorrhizal fungi (AMF), including Glomus versiforme and G. intraradices, the regulation of phosphate transporters that are expressed, typically upregulated under P deficiency but their role in P transfer to the host plant have not been characterized.^5,6P. indica was reported to be involved in high salt tolerance, disease resistance and strong growth-promoting activities leading to enhancement of host plant yield.^7–9 Recently, we have shown the role of PiPT in the P transport to the host plant.¹⁰ Here we discuss the performance of P. indica (grown under P-rich and -deprived conditions and colonized with the host plant) and its involvement in the P transportation to, and the growth of the host plant.     

Endophyte-induced Verticillium protection in tomato is range-restricted

Endophytes, bacterial, fungal or viral, colonize plants often without causing visible symptoms. More important, they may benefit host plants in many ways, most notably by preventing diseases caused by normally virulent pathogens. Previous studies have shown that an isolate of V. dahliae from eggplant, Dvd-E6, can colonize tomato endophytically, producing taller and more robust tomato plants while providing protection against a virulent V. dahliae, race 1 (Vd1) isolate. Expression analyses suggest this requires interplay between Dvd-E6 and the plant that involves resistance and defense genes. To examine the possibility of a broader effect, dual interactions have been further examined with a more distantly related pathogen, Verticillium albo-atrum (Vaa). The results indicate Dvd-E6 colonization selectively modifies the expression of specific tomato genes to be detrimental to Vd1 but not Vaa, providing evidence that Verticillium-induced protection is range-restricted.Key words: cross-protection, endophyte, lycopersicon, tolerance, VerticilliumAn “endophyte” commonly is defined as a “fungus or bacterium living within plants without causing visible symptoms of disease”; a “pathogen” is regarded as “a disease causing biological agent”. Historically, plant biologists have tended to consider these as two very different and distinct classes of organisms but accumulating evidence now suggests that the boundaries between mutualism and parasitism are not as defined as previously thought. Many organisms can occupy both ecological niches¹ depending on the genotype of the host, the genotype of the organism itself and interaction with the environment. Indeed, this “dual life style” may be a significant factor in the evolutionary dynamics of pathogen resistance, tolerance and susceptibility.²One of the more recent additions to the growing list of dual life style (endophyte/pathogen) fungi is Verticillium dahliae,³ a causal agent of vascular wilt disease or early dying syndrome in a broad range of the plant species.⁴ When Verticillium infects a plant three different host responses can occur: resistance, susceptibility or tolerance. The phenomenon of tolerance has been associated with Verticillium spp. for decades but research on mechanisms governing the development of the plant/Verticillium interaction has focused on the compatible and incompatible interactions and little is known about the tolerant state (reviewed in ref. ⁵). In a recent series of papers we have identified an isolate of Verticillium dahliae, known as Dvd-E6, that colonizes tomatoes, cv Craigella, resulting in a stable tolerant condition, that we have used as a model system to investigate the biological and molecular bases of plant tolerance to Verticillium spp. The Craigella/Dvd E6 interaction has a number of interesting but unanticipated properties. Host plants tend to be taller and more robust than their uninfected counterparts⁶ and colonization of Craigella by Dvd-E6 provides protection against its virulent Verticillium dahliae race 1 (Vd1) cousin, limiting both Vd1 colonization and symptom development during dual infections.⁷ Such attributes normally are associated with plant/endophyte partnerships, strongly suggesting that under some conditions, Verticillium dahliae can assume an endophytic role. Apparently interplay between Dvd-E6 and the plant establishes protection against the virulent Vd1.⁷Many endophytic infections provide protection for the host against a broad range of pathogens.¹ To test the ability of Dvd-E6 infection to protect Craigella against a more distantly related Verticillium pathogen we have now examined dual interactions with Verticillium albo-atrum (Vaa). Susceptible Craigella⁸ seedlings again were inoculated at the 4-leaf stage by dipping the roots in Vd1, Dvd-E6 or Vaa conidial suspensions (1 × 10⁷ spores/ml in 0.5% gelatin solution) to establish homogeneous interactions (reviewed in ref. ⁷). For dual interactions, seedlings were inoculated with Dvd-E6 spores at the 3-leaf stage and reinoculated at the 4-leaf stage with either Vd1 or Vaa spore suspension to establish the mixed infections. Control seedlings were root dipped in gelatin solution alone. Plants were scored for symptom expression on a 0 (no symptoms) to 5 (plant dead) scale as described by Shittu and co-workers (2009) and the top two-thirds of the stems harvested at 5 and 10 days post inoculation (d.p.i.) for extraction and fungal DNA assays.^7,9All experimental results are summarized in Figure 1. At 10 d.p.i., the disease scores for Dvd-E6-infected plants were approximately one-third that of the Vd1-or Vaa-infected plants. More interesting were the symptoms in the dual interactions, Dvd-E6/Vd1 mimicking the tolerant plants with low disease and Dvd-E6/Vaa-infected plants exhibiting the highest disease scores, similar to Vd1-or Vaa-infected plants. When the amount of fungal DNA in the stems was assessed, the total fungal biomass in both dual infections as well as the Dvd-E6 and Vd1-infected plants at 5 and 10 dpi was similar (light and dark gray bars), the amount of Vaa being somewhat lower. In the mixed infections, however, most of the DNA (>90%) was of Dvd-E6 origin (white bars). More important, the Vd1 DNA level in Dvd-E6/Vd1 plants was substantially reduced relative to plants infected with Vd1 alone while the Vaa DNA levels stayed about the same in the single and double infections. This indicates that Vd1 colonization is restricted⁷ by the presence of Dvd-E6 while Vaa is not.Open in a separate windowFigure 1Comparison of symptoms and levels of V. dahliae 1 or V. albo-atrum DNA in susceptible Craigella tomato simultaneously infected with V. dahliae Dvd-E6. Individual (E6, Vd1 and Vaa) and mixed (E6/Vd1 and E6/Vaa) infections were established as previously described.⁷ Plants were scored (upper) at 5 (light gray bars) and 10 (dark gray bars) dpi for symptoms (i.e., disease scores ± SD) or assayed⁷ for total levels of Verticillium DNA (i.e., ng/g plant tissue ± SD) in the stems (lower). In mixed infections levels for each fungus also were determined (black and white bars, respectively). Results summarize the data for 9–12 plants per time point for each interaction.These results demonstrate that Dvd-E6 infection protects Craigella tomatoes against colonization by and symptom development from Vd1 but not Vaa. Past studies often have suggested that the protective effect stems from an endophyte-induced activation of systemic acquired resistance (SAR) in the host providing protection against a broad range of pathogens.¹⁰ However, in the tolerant CS//Dvd-E6 interaction the protective effect appears to be targeted more directly, allowing Dvd-E6 to effectively restrict it''s virulent cousin, Vd1. In this context, it may be important that both of the Verticillium dahliae isolates from tomato are endemic to Ontario^7,11 and potentially in direct competition, while Verticillium albo-atrum from tomato is not. Previous molecular analyses suggested that the protective effect provided by Dvd-E6 colonization requires a genetic interplay with the host, selectively modifying the expression of specific tomato genes to be detrimental to Vd1. The experimental results presented here provide evidence that Verticillium-induced protection is restricted in range.     

Root tips moving through soil: An intrinsic vulnerability

Root elongation occurs by the generation of new cells from meristematic tissue within the apical 1–2 mm region of root tips. Therefore penetration of the soil environment is carried out by newly synthesized plant tissue, whose cells are inherently vulnerable to invasion by pathogens. This conundrum, on its face, would seem to reflect an intolerable risk to the successful establishment of root systems needed for plant life. Yet root tip regions housing the meristematic tissues repeatedly have been found to be free of microbial infection and colonization. Even when spore germination, chemotaxis, and/or growth of pathogens are stimulated by signals from the root tip, the underlying root tissue can escape invasion. Recent insights into the functions of root border cells, and the regulation of their production by transient exposure to external signals, may shed light on long-standing observations.Key words: border cells, chemotaxis, zoospores, neutrophil extracellular traps (NETs)…The evidence suggests that there has evolved within plants, mechanisms for extremely rapid adjustment to changes in the soil environment. The logical conclusion is that plants can and do selectively manipulate the ecological balances within the rhizosphere to their own advantage.¹“Sloughed root cap cells” that detach from the root tip were long presumed to be moribund tissue serving to lubricate passage of the elongating root.² The discovery nearly a century ago that these cells from Zea mays L. and Pisum sativum L. can remain 100% viable for weeks after detachment into hydroponic culture did not alter this perception.³ In recent decades, studies have shown that the cells from root caps of most species are metabolically active and can survive even after detachment into the soil.⁴ Moreover, the cell populations express distinct patterns of gene expression reflecting tissue specialization and were therefore given the name root ‘border’ cells.⁵ Like ‘border towns’ that exist at the boundary of disparate countries and cultures, border cells are part of the plant and part of the soil, yet distinct from both.The soil is a dynamic environment whose pH, surface charge, water availability, texture and composition can range markedly on a large and small scale.^1,6,7 The concept of a ‘microniche’ emphasizes that the biological requirements for a particular soil microorganism may be met within one site but not another site only a micron away.⁸ Thus, the rhizosphere—the region adjacent to root surfaces—can support much higher levels of microorganisms than bulk soil a few millimeters distant.⁹ This phenomenon is recognized to be driven by an increased availability of nutrients released from plants into the external environment.¹⁰ Less well recognized is the dynamic variation that occurs along the root surface, and its significance in patterns of disease development. As roots emerge and the new tissue differentiates progressively through stages from root cap, root apical meristem, elongation zone, and finally mature roots with lignified cell walls, the material released into the environment also changes.^11–13 More than 90% of bulk carbon released from young roots of legumes is delivered by the root cap, a 1 mm zone at the apex.¹⁴ Some pathogens are attracted specifically to the root tip region, presumably in response to such exudates.^15,16 For example, instantaneous swarming occurs when a cotton root is placed into a suspension of Pythium dissotocum zoospores (Sup. Fig. 1). This host-specific attraction is specific to the root tip region where border cells are present (Sup. Fig. 2). Border cells remain attractive to zoospores when removed from the root (Sup. Fig. 3). The nature of the attractant is not known, but its impact is localized and transient (Sup. Fig. 4).Newly generated tissue is highly susceptible to infection by pathogens, in general, so elongating root tips would be predicted to be vulnerable to invasion. And yet, root apices repeatedly have been found to escape infection and colonization.^17–19 Recent discoveries about parallels between mammalian white blood cells and root border cells may provide new insight into this apparent conundrum.²⁰ Neutrophils, a type of white blood cell, are produced in response to infection. Neutrophil extracellular traps (NETs) then attract and kill the invader through a process that requires extracellular DNA (exDNA) and an array of extracellular proteins.^21,22 Border cell production, like that of neutrophils, also is induced in response to signals from pathogens and root tip resistance to infection requires exDNA and an array of extracellular proteins.^20,23 Root tip specific chemotaxis, like that seen with Pythium zoospores, has been presumed to involve steps in a process of pathogen invasion.^15,16 It may, instead, involve a process of extracellular trapping and killing by cells designed to protect root meristems from invasion, in a manner analogous to that which occurs in mammalian defense. If tests confirm this model, the mystery of how root tips escape infection by soilborne pathogens they attract could be resolved.