Detection and identification of bacteria intimately associated with fungi of the order Sebacinales

Because of their beneficial impact on plants, the highly diverse mycorrhizal fungi grouped in the order Sebacinales lay claim to high ecological and agricultural significance. Here, we describe for the first time associations of Sebacinoid members with bacteria. Using quantitative PCR, denaturating gradient gel electrophoresis and fluorescence in situ hybridization, we detected an intimate association between Piriformospora indica and Rhizobium radiobacter, an alpha-Proteobacterium. The stability of the association, vertical transmission of the bacteria during asexual fungal reproduction and fungal plant colonization was monitored using R. radiobacter-specific primers. Treatment of mycelium or fungal protoplasts with antibiotics highly efficient against the free bacteria failed to cure the fungus. Barley seedlings dip-inoculated with R. radiobacter showed growth promotion and systemic resistance to the powdery mildew fungus Blumeria graminis comparable to P. indica inoculation. By screening additional isolates of the Sebacina vermifera complex, three species-specific associations with bacteria from the genera Paenibacillus, Acinetobacter and Rhodococcus were found. These findings suggest that Sebacinales species regularly undergo complex interactions involving host plants and bacteria reminiscent of other ectomycorrhizal and endomycorrhizal associations.     

Growth promotion of Chinese cabbage and Arabidopsis by Piriformospora indica is not stimulated by mycelium-synthesized auxin

Piriformospora indica, an endophytic fungus of the order Sebacinales, interacts with the roots of a large variety of plant species. We compared the interaction of this fungus with Chinese cabbage (Brassica campestris subsp. chinensis) and Arabidopsis seedlings. The development of shoots and roots of Chinese cabbage seedlings was strongly promoted by P. indica and the fresh weight of the seedlings increased approximately twofold. The strong stimulation of root hair development resulted in a bushy root phenotype. The auxin level in the infected Chinese cabbage roots was twofold higher compared with the uncolonized controls. Three classes of auxin-related genes, which were upregulated by P. indica in Chinese cabbage roots, were isolated from a double-subtractive expressed sequence tag library: genes for proteins related to cell wall acidification, intercellular auxin transport carrier proteins such as AUX1, and auxin signal proteins. Overexpression of B. campestris BcAUX1 in Arabidopsis strongly promoted growth and biomass production of Arabidopsis seedlings and plants; the roots were highly branched but not bushy when compared with colonized Chinese cabbage roots. This suggests that BcAUX1 is a target of P. indica in Chinese cabbage. P. indica also promoted growth of Arabidopsis seedlings but the auxin levels were not higher and auxin genes were not upregulated, implying that auxin signaling is a more important target of P. indica in Chinese cabbage than in Arabidopsis. The fungus also stimulated growth of Arabidopsis aux1 and aux1/axr4 and rhd6 seedlings. Furthermore, a component in an exudate fraction from P. indica but not auxin stimulated growth of Chinese cabbage and Arabidopsis seedlings. We propose that activation of auxin biosynthesis and signaling in the roots might be the cause for the P. indica-mediated growth phenotype in Chinese cabbage.     

Ethylene supports colonization of plant roots by the mutualistic fungus Piriformospora indica

The mutualistic basidiomycete Piriformospora indica colonizes roots of mono- and dicotyledonous plants, and thereby improves plant health and yield. Given the capability of P. indica to colonize a broad range of hosts, it must be anticipated that the fungus has evolved efficient strategies to overcome plant immunity and to establish a proper environment for nutrient acquisition and reproduction. Global gene expression studies in barley identified various ethylene synthesis and signaling components that were differentially regulated in P. indica-colonized roots. Based on these findings we examined the impact of ethylene in the symbiotic association. The data presented here suggest that P. indica induces ethylene synthesis in barley and Arabidopsis roots during colonization. Moreover, impaired ethylene signaling resulted in reduced root colonization, Arabidopsis mutants exhibiting constitutive ethylene signaling, -synthesis or ethylene-related defense were hyper-susceptible to P. indica. Our data suggest that ethylene signaling is required for symbiotic root colonization by P. indica.     

Salt tolerance of barley induced by the root endophyte Piriformospora indica is associated with a strong increase in antioxidants

The root endophytic basidiomycete Piriformospora indica has been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. Biochemical mechanisms underlying P. indica-mediated salt tolerance were studied in barley (Hordeum vulgare) with special focus on antioxidants. Physiological markers for salt stress, such as metabolic activity, fatty acid composition, lipid peroxidation, ascorbate concentration and activities of catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase and glutathione reductase enzymes were assessed. Root colonization by P. indica increased plant growth and attenuated the NaCl-induced lipid peroxidation, metabolic heat efflux and fatty acid desaturation in leaves of the salt-sensitive barley cultivar Ingrid. The endophyte significantly elevated the amount of ascorbic acid and increased the activities of antioxidant enzymes in barley roots under salt stress conditions. Likewise, a sustained up-regulation of the antioxidative system was demonstrated in NaCl-treated roots of the salt-tolerant barley cultivar California Mariout, irrespective of plant colonization by P. indica. These findings suggest that antioxidants might play a role in both inherited and endophyte-mediated plant tolerance to salinity.     

Piriformospora indica-a mutualistic basidiomycete with an exceptionally large plant host range

Piriformospora indica is a basidiomycete of the order Sebacinales, representing a model for the study of mutualistic symbiosis and, beyond that, the plant immune system. The fungus colonizes the roots of a wide range of vascular plants, increasing their growth, seed yield and adaptation to abiotic and biotic stresses. The fungal colonization of roots begins with a biotrophic growth phase, in which living cells are colonized, and continues with a cell death-dependent phase, in which root cells are actively killed by the fungus. The complexity of sebacinalean symbiosis is further enhanced by the presence of endocellular bacteria which may represent significant determinants for a successful outcome of the symbioses. Molecular ecological analyses have revealed an exceptional relevance of sebacinoid fungi in natural ecosystems worldwide. This natural competence could be rooted in their phenotypic adaptability, which, for instance, allows P. indica to grow readily on various synthetic media and to colonize distinct hosts. In molecular and genetic studies, P. indica's mutualistic colonization strategy has been partly unravelled, showing that the jasmonate pathway is exploited for immune suppression and successful development in roots. Research on P. indica supports efforts to make the bioprotective potential of the fungus accessible for agricultural plant production. The decoding of P. indica's genome has revealed its potential for application as bioagent and for targeted improvement of crop plants in biotechnology-based approaches.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant–microbe interactions and involves early plant protein modifications in the endoplasmic reticulum and at the plasma membrane

Piriformospora indica , an endophytic fungus of the Sebacinaceae family, colonizes the roots of a wide variety of plant species and promotes their growth, in a manner similar to arbuscular mycorrhizal fungi. The results of the present study demonstrate that the fungus interacts also with the non-mycorrhizal host Arabidopsis thaliana and promotes its growth. The interaction is detectable by the appearance of a strong autofluorescence in the roots, followed by the colonization of root cells by fungal hyphae and the generation of chlamydospores. Promotion of root growth was detectable even before noticeable root colonization. Membrane-associated proteins from control roots and roots after cultivation with P. indica were separated by two-dimensional gel-electrophoresis and identified by electrospray ionization mass spectrometry and tandem mass spectrometry. Differences were found in the expression of glucosidase II, beta-glucosidase PYK10, two glutathione- S -transferases and several so-far uncharacterized proteins. Based on conserved domains present in the latter proteins their possible roles in plant–microbe interaction are predicted. Taken together, the present results suggest that the interaction of Arabidopsis thaliana with P. indica is a powerful model system to study beneficial plant–microbe interaction at the molecular level. Furthermore, the successful accommodation of the fungus in the root cells is preceded by protein modifications in the endoplasmatic reticulum as well as at the plasma membrane of the host.     

Seed Treatment of Barley with Idriella bolleyi causes Systemically Enhanced Defence against Root and Leaf Infection by Bipolaris sorokiniana

Inoculation of barley seed with the saprophytic fungus Idriella bolleyi caused systemically improved resistance on both leaves and roots of young plants to subsequent infection with the necrotrophic fungus Bipolaris sorokiniana . Lesion sizes were reduced by up to 50% as a result of the seed treatment. Seed and root inoculation with I. bolleyi induced biochemical defence responses in the plants as shown by slight accumulation of pathogenesis-related (PR) proteins, but the accumulation was not as great as when the roots were inoculated with B. sorokinina . Conidia of I. bolleyi applied to seed survived well during storage, with no significant reduction in colony forming units (CFUs) occurring at 15°C after 2 months. However, storage at 28°C decreased the viability of the applied conidia. The fungus colonized the seed and roots during field conditions as indicated by high numbers of CFUs. Two months after sowing, frequencies of I. bolleyi were higher on plants treated with the fungus than on control plants where colonization occurred naturally from field soil. The implications of the results are discussed in relation to earlier reports of biological control of cereal root diseases by I. bolleyi and induced resistance.     

Colonisation of barley roots by endophytic Fusarium equiseti and Pochonia chlamydosporia: Effects on plant growth and disease

Colonisation of plant roots by endophytic fungi may confer benefits to the host such as protection against abiotic or biotic stresses or plant growth promotion. The exploitation of these properties is of great relevance at an applied level, either to increase yields of agricultural crops or in reforestation activities. Fusarium equiseti is a naturally occurring endophyte in vegetation under stress in Mediterranean ecosystems. Pochonia chlamydosporia is a nematode egg-parasitic fungus with a worldwide distribution. Both fungi have the capacity to colonise roots of non-host plants endophytically and to protect them against phytopathogenic fungi under laboratory conditions. The aim of this study was to evaluate the root population dynamics of these fungi under non-axenic practical conditions. Both fungal species were inoculated into barley roots. Their presence in roots and effects on plant growth and incidence of disease caused by the pathogen Gaeumannomyces graminis var. tritici were monitored periodically. Both fungi colonised barley roots endophytically over the duration of the experiment and competed with other existing fungal root colonisers. Furthermore, colonisation of roots by P. chlamydosporia promoted plant growth. Although a clear suppressive effect on disease could not be detected, F. equiseti isolates reduced the mean root lesion length caused by the pathogen. Results of this work suggest that both F. equiseti and P. chlamydosporia are long-term root endophytes that confer beneficial effects to the host plant.     

The role of auxins and cytokinins in the mutualistic interaction between Arabidopsis and Piriformospora indica

Arabidopsis growth and reproduction are stimulated by the endophytic fungus Piriformospora indica. The fungus produces low amounts of auxins, but the auxin levels and the expression of auxin-regulated genes are not altered in colonized roots. Also, mutants with reduced auxin levels (ilr1-1, nit1-3, tfl2, cyp79 b2b3) respond to P. indica. However, the fungus rescues the dwarf phenotype of the auxin overproducer sur1-1 by converting free auxin into conjugates, which also results in the downregulation of the auxin-induced IAA6 and the upregulation of the P. indica-induced LRR1 gene. The fungus produces relatively high levels of cytokinins, and the cytokinin levels are higher in colonized roots compared with the uncolonized controls. trans-Zeatin cytokinin biosynthesis and the CRE1/AHK2 receptor combination are crucial for P. indica-mediated growth stimulation, while mutants lacking cis-zeatin, impaired in other cytokinin receptor combinations, or containing reduced cytokinin levels respond to the fungus. Since root colonization is not affected in the cytokinin mutants, we propose that cytokinins are required for P. indica-induced growth promotion. Finally, a comparative analysis of the phytohormone mutants allows the conclusion that the response to P. indica is independent of the architecture and size of the roots.     

印度梨形孢促进蒺藜苜蓿生长及其提高耐盐性研究

【目的】研究盐胁迫下印度梨形孢定殖对豆科模式植物蒺藜苜蓿生长发育的影响。【方法】通过分析不同生境下植物的根长、根鲜重和茎鲜重,以及体内抗氧化物酶活性、脯氨酸含量、甜菜碱醛脱氢酶基因(BADH)的表达,确定印度梨形孢对蒺藜苜蓿生长的促进作用,并初步阐释印度梨形孢诱导植物耐盐性的机制。【结果】印度梨形孢能在蒺藜苜蓿根部定殖并能促进植物的生长发育,有效缓解盐胁迫造成的生长抑制。印度梨形孢能提高植物体内抗氧化物酶活性,增加游离脯氨酸含量并诱导BADH基因的表达。【结论】印度梨形孢作为植物生长促进因子可以用来提高植物耐盐性,实现盐碱土壤的间接改良。     

Colonization of barley (Hordeum vulgare) with Salmonella enterica and Listeria spp

Colonization of barley plants by the food-borne pathogens Salmonella enterica serovar typhimurium and three Listeria spp. (L. monocytogenes, L. ivanovii, L. innocua) was investigated in a monoxenic system. Herbaspirillum sp. N3 was used as a positive control and Escherichia coli HB101 as a negative control for endophytic root colonization. Colonization of the plants was tested 1-4 weeks after inoculation by determination of CFU, specific PCR assays and fluorescence in situ hybridization (FISH) with fluorescently labelled oligonucleotide probes in combination with confocal laser scanning microscopy (CLSM). Both S. enterica strains were found as endophytic colonizers of barley roots and reached up to 2.3 x 10(6) CFU per g root fresh weight after surface sterilization. The three Listeria strains had 10-fold fewer cell numbers after surface sterilization on the roots and therefore were similar to the results of nonendophytic colonizers, such as E. coli HB101. The FISH/CSLM approach demonstrated not only high-density colonization of the root hairs and the root surface by S. enterica but also a spreading to subjacent rhizodermis layers and the inner root cortex. By contrast, the inoculated Listeria spp. colonized the root hair zone but did not colonize other parts of the root surface. Endophytic colonization of Listeria spp. was not observed. Finally, a systemic spreading of S. enterica to the plant shoot (stems and leaves) was demonstrated using a specific PCR analysis and plate count technique.