Root colonization by Piriformospora indica enhances grain yield in barley under diverse nutrient regimes by accelerating plant development

The basidiomycete fungus Piriformospora indica colonizes roots of a broad range of mono- and dicotyledonous plants. It confers enhanced growth, improves resistance against biotic and tolerance to abiotic stress, and enhances grain yield in barley. To analyze mechanisms underlying P. indica-induced improved grain yield in a crop plant, the influence of different soil nutrient levels and enhanced biotic stress were tested under outdoor conditions. Higher grain yield was induced by the fungus independent of different phosphate and nitrogen fertilization levels. In plants challenged with the root rot-causing fungus Fusarium graminearum, P. indica was able to induce a similar magnitude of yield increase as in unchallenged plants. 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. On the other hand, barley plants colonised with the endophyte developed faster, and were characterized by a higher photosynthetic activity at low light intensities. Together with the increased root formation early in development these factors contribute to faster development of ears as well as the production of more tillers per plant. The results indicate that the positive effect of P. indica on grain yield is due to accelerated growth of barley plants early in development, while improved phosphate supply—a central mechanism of host plant fortification by arbuscular mycorrhizal fungi—was not observed in the P. indica-barley symbiosis.     

Unraveling the role of fungal symbionts in plant abiotic stress tolerance

Fungal symbionts have been found to be associated with every plant studied in the natural ecosystem, where they colonize and reside entirely or partially in the internal tissues of their host plant. Fungal endophytes can express/form a range of different lifestyle/relationships with different host including symbiotic, mutualistic, commensalistic and parasitic in response to host genotype and environmental factors. In mutualistic association fungal endophyte can enhance growth, increase reproductive success and confer biotic and abiotic stress tolerance to its host plant. Since abiotic stress such as, drought, high soil salinity, heat, cold, oxidative stress and heavy metal toxicity is the common adverse environmental conditions that affect and limit crop productivity worldwide. It may be a promising alternative strategy to exploit fungal endophytes to overcome the limitations to crop production brought by abiotic stress. There is an increasing interest in developing the potential biotechnological applications of fungal endophytes for improving plant stress tolerance and sustainable production of food crops. Here we have described the fungal symbioses, fungal symbionts and their role in abiotic stress tolerance. A putative mechanism of stress tolerance by symbionts has also been covered.Key words: abiotic stress, endophytes, fungal symbiont, mycorrhizal fungus, Piriformospora indica, stress tolerance, symbiosis     

温郁金内生真菌Chaetomium globosum L18对植物病原菌的抑菌谱及拮抗机理

通过体外培养法,研究了药用植物温郁金内生真菌Chaetomium globosum L18对几种常见的植物病原菌的抑菌谱及其拮抗机理。结果表明,Chaetomium globosum L18对多种植物病原真菌和细菌均有不同程度的抑制作用,具有较广的抑菌谱,但对不同植物病原菌的抑制作用具有显著性差异(P<0.05),抑制率最高可达到92.9%;抑菌机制结果显示,竞争作用和重寄生作用是其主要的拮抗机制之一;发酵产物抑制作用测定发现,内生真菌Chaetomium globosum L18能够分泌产生抗菌物质抑制病原菌菌丝的生长和孢子萌发,可引起病原菌菌丝菌丝膨大成串珠状,分枝增多,分枝顶端膨胀后细胞壁破裂,原生质外溢,产生溶菌作用;使分生孢子萌发畸形,萌发率降低。     

Endophytic Fungal Flora from Roots and Fruits of an Indian Neem Plant <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss., and Impact of Culture Media on their Isolation

Azadirachta indica A. Juss. (neem), native to India, is well known worldwide for its insecticidal and ethanopharmacological properties. Although endophytic microbes are known from this plant as only leaves and stems were the subjects of past reports. Now, a variety of procedures and a number of different media were used to isolate the maximum number of endophytic fungi from unripe fruits and roots. A total of 272 isolates of 29 filamentous fungal taxa were isolated at rate of 68.0% from 400 samples of three different individual trees (at locations-Az1, Az2, Az3). Mycological agar (MCA) medium yielded the highest number of isolates (95, with a 14.50% isolation rate) with the greatest species richness. Mycelia Sterilia (1, 2, 3) accounted for 11.06%, Coelomycetes 7.25%, while Hyphomycetes showed the maximum number of representative isolates (81.69%). Mycelia-Sterilia (1, 2, 3), based on their 5.8S ITS 1, ITS2 and partial 18S and 28S rDNA sequences were identified as Fusarium solani (99%), Chaetomium globosum (93%) and Chaetomium globosum (93%) respectively. Humicola, Drechslera, Colletotrichum, and Scytalidium sp. were some of the peculiar fungal endophytes recovered from this plant.     

In vitro plant development and root colonization of Coleus forskohlii by Piriformospora indica

The present study was conducted for optimization of in vitro substrates under aseptic conditions for interaction of Piriformospora indica with the medicinal plant Coleus forskohlii. It aims to test the effects of different substrates on P. indica colonization as well as growth parameters of the in vitro raised C. forskohlii. Interaction of in vitro C. forskohlii with root endophyte P. indica under aseptic condition resulted in increase in growth parameters in fungus colonized plants. It was observed that P. indica promoted the plant’s growth in all irrespective of substrates used for co-culture study. The growth was found inferior in liquid compared to semisolid medium as well as there was problem of hyperhydricity in liquid medium. P. indica treated in vitro plantlets were better adapted for establishment under green house compared to the non treated plants due to fungal intervention.     

Controlling Myzus persicae with recombinant endophytic fungi Chaetomium globosum expressing Pinellia ternata agglutinin: using recombinant endophytic fungi to control aphids

Aims: Sap‐sucking insect pests have become the major threats to many crops in recent years; however, only a few biopesticides have been developed for controlling those pests. Here, we developed a novel pest management strategy, which uses endophytes to express anti‐pest plant lectins. Methods and Results: The fungal endophyte of Chaetomium globosum YY‐11 with anti‐fungal activities was isolated from rape seedlings. Pinellia ternata agglutinin (pta) gene was cloned into YY‐11 mediated by Agrobacterium tumefaciens. The positive transformants, as selected by antibiotic resistance, were evaluated using PCR and Western blot assay. We found that the recombinant endophytes colonized most of the crops, and the resistance of rape inoculated with recombinant endophytic fungi significantly inhibited the growth and reproduction of Myzus persicae. Conclusions: Our results showed that the recombinant endophytes expressing Pinellia ernata agglutinin (PTA) may endow hosts with resistance against sap‐sucking pests. Significance and Impact of the Study: This research may have important implications for using endophytes to deliver insecticidal plant lectin proteins to control sap‐sucking pests for crop protection.     

Intracellular targeting of ascomycetous catalase-peroxidases (KatG1s)

Bifunctional catalase-peroxidases (KatGs) are heme oxidoreductases widely spread among bacteria, archaea and among lower eukaryotes. In fungi, two KatG groups with different localization have evolved, intracellular (KatG1) and extracellular (KatG2) proteins. Here, the cloning, expression analysis and subcellular localization of two novel katG1 genes from the soil fungi Chaetomium globosum and Chaetomium cochliodes are reported. Whereas, the metalloenzyme from Ch. globosum is expressed constitutively, Ch. cochliodes KatG1 reveals a slight increase in expression after induction of oxidative stress by cadmium ions and hydrogen peroxide. The intronless open reading frames of both Sordariomycetes katG1 genes as well as of almost all fungal katG1s possess two peroxisomal targeting signals (PTS1 and PTS2). Peroxisomal localization of intracellular eukaryotic catalase-peroxidases was verified by organelle separation and immunofluorescence microscopy. Co-localization with the peroxisomal enzyme 3-ketoacyl-CoA-thiolase was demonstrated for KatGs from Magnaporthe grisea, Chaetomium globosum and Chaetomium cochliodes. The physiological role of fungal catalase-peroxidases is discussed.     

The plant strengthening root endophyte Piriformospora indica: potential application and the biology behind

The successful conversion of plant production systems from conventional resource-exhausting to sustainable strategies depends on knowledge-based management of environmental factors. Root-inhabiting fungi came more and more into focus because their hyphae connect in ideal manner resources and challenges of the surrounding with the plant. A paradigm for such root endophytes is presented by the basidiomycete Piriformospora indica. This fungus possesses a broad host spectrum and positively affects different aspects of plant performance. This so far unique combination of attributes makes P. indica and its close relatives among the Sebacinales very interesting tools for cultivation of various crops. This review will outline the different aspects required to apply this root endophyte in agri- and horticulture concerning plant growth, plant nutrition and plant defence or tolerance thereby explaining what is known about the biological basis for the observed effects. Open questions and challenges for successful inoculum production and application will be discussed.     

Exogenous auxin affects the oxidative burst in barley roots colonized by Piriformospora indica

Beside a cardinal role in coordination of many developmental processes in the plant, the phytohormone auxin has been recognized as a regulator of plant defense. The molecular mechanisms involved are still largely unknown. Using a sensitive chemiluminescence assay, which measures the oxidation of luminol in the presence of H₂O₂ by horseradish peroxidase (HRP), we report here on the ability of exogenously added indole-3-acetic acid (IAA) to enhance the suppressive effect of the root endophyte Piriformospora indica on the chitin-elicited oxidative burst in barley roots. Thus, the potential of P. indica to produce free IAA during the early colonization phase in barley might provide the symbiont with a means to interfere with the microbe-associated molecular patterns (MAMP)-triggered immunity.     

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.     

Occurrence of fungal endophytes in cultivars ofTriticum aestivum in South Africa

Fungal endophytes were isolated from leaves, roots and stems of four wheat cultivars and a breeding line at three different sampling dates during the 1993 growing season. Of the 55 different fungal taxa encountered, 19 were present at relative importance values of more than 5%. No cultivar-related differences in the assembleges of endophytes were observed.Phoma glomerata was not restricted to only one tissue type, whereasAlternaria alternata, basidiomycete sp. 1,Pleospora herbarum andEpicoccum nigrum occurred primarily in the leaves, andFusarium avenaceum was extremely frequent in roots. In general, colonization by endophytes increased with the age of the plants. Most endophytes were isolated from wheat leaves. Successional colonization of a given tissue type was quantitative rather than qualitative, with a given fungal taxon increasing or decreasing over the period sampled, rather than replacing the fungi initially encountered.Present address: Téra d'Sott 5, CH-6949, Comano, Switzerland.     

The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

Role of Diverse Non-Systemic Fungal Endophytes in Plant Performance and Response to Stress: Progress and Approaches

Plant–fungal symbiotic associations are ubiquitously distributed in natural plant communities. Besides the well-studied mycorrhizal symbiosis and grass systemic clavicipitaceous endophytes, recently, nonsystemic and horizontally transmitted fungal endophytes serving as plant symbionts have been increasingly recognized. Pure culture isolation and culture-independent molecular methods indicate that all parts of healthy plant tissues potentially harbor diverse and previously unknown fungal lineages. Limited evidence also supports a hypothesis that endophytic mycobiota dynamics may have a role in evolution of plants. High variability or “balanced antagonism” can be generally characterized with host–endophyte interactions, which implies that the outcome of symbiotic interactions can fall within a continuum ranging from mutualism to commensalism, and ultimately pathogenicity. Despite this complicated system, admittedly, fungal endophytes really endow the host with an extended phenotype. Accumulating facts illustrate that plant nutrition acquisition, metabolism, and stress tolerance may be strengthened or modulated via fungal symbionts. Piriformospora indica, a member of the order Sebacinales, simultaneously confers host resistance to biotic and abiotic stress. The ecological relevance of other fungal groups, including foliar endophytes, root dark septate endophytes (DSEs), some opportunistic and avirulent microsymbionts (for example, Trichoderma and Fusarium), and even uncultured fungi structurally and physiologically integrated with host tissues, are also being deeply exploited. Production of bioactive metabolites by fungi, overexpression of stress-related enzymes, and induced resistance in hosts upon fungal colonization are responsible for direct or indirect beneficial effects to hosts. More knowledge of endophyte-mediated enhancement of host performance and fitness will offer alternatively valuable strategies for plant cultivation and breeding. Meanwhile, with unprecedented loss of biodiversity, discovery of indigenously novel symbiotic endophytes from natural habitats is urgently needed. In addition, we present some approaches and suggestions for studying host–endophyte interactions.     

The root endophyte fungus Piriformospora indica leads to early flowering,higher biomass and altered secondary metabolites of the medicinal plant,Coleus forskohlii

This study was undertaken to investigate the influence of plant probiotic fungus Piriformospora indica on the medicinal plant C. forskohlii. Interaction of the C. forskohlii with the root endophyte P. indica under field conditions, results in an overall increase in aerial biomass, chlorophyll contents and phosphorus acquisition. The fungus also promoted inflorescence development, consequently the amount of p-cymene in the inflorescence increased. Growth of the root thickness was reduced in P. indica treated plants as they became fibrous, but developed more lateral roots. Because of the smaller root biomass, the content of forskolin was decreased. The symbiotic interaction of C. forskohlii with P. indica under field conditions promoted biomass production of the aerial parts of the plant including flower development. The plant aerial parts are important source of metabolites for medicinal application. Therefore we suggest that the use of the root endophyte fungus P. indica in sustainable agriculture will enhance the medicinally important chemical production.     

Endophytic fungus Serendipita indica increased nutrition absorption and biomass accumulation in Cunninghamia lanceolata seedlings under low phosphate

Cunninghamia lanceolata is important forest tree species in southern China, and its successive plantations resulted in degradation of soil fertility in pure stands, causing decline in forest productivity. How to improve productivity in C. lanceolata pure stands is a tough task. Usage of mycorrhizal fungi might be a plausible access to the task. The objective is to study the possibility of the endophytic fungus Serendipita indica (named formerly as Piriformospora indica) in culture of C. lanceolata. Seeds were sowed in plastic pots with river sand. When seedlings had two true leaves, hyphae suspension solution of S. indica was added to near the roots of seedlings in each plastic pot. Such pots with seedlings were placed in a greenhouse and normal management was carried out for the seedlings. Symbiosis effects on root development, nutrition uptake and allocation, and biomass accumulation of C. lanceolata seedlings under low phosphate were investigated. The results showed that S. indica could symbiose with C. lanceolata. The symbiosis did not result in significant changes in root system architecture under low phosphate, but significantly increased nitrogen and phosphorus levels in leaves under low phosphate. Although the symbiosis did not significantly increased nitrogen allocation in leaves under low phosphate, it significantly increased phosphorus allocation in leaves. The interaction between S. indica and C. lanceolata resulted in increase in total biomass under low phosphate and changes in biomass allocation between shoots and roots. The results suggested that S. indica helps host plants to absorb more nutrients under low phosphate and to allocate more nitrogen and phosphate to leaves, promoting plant growth; the fungus might be used in pure stands of C. lanceolata because of its large-scaled axenic culture.     

Experimentation on Degradation of Petroleum in Contaminated Soils in the Root Zone of Maize (Zea Mays L.) Inoculated with Piriformospora Indica

Plant-based methods such as rhizodegradation are very promising for the remediation of petroleum-contaminated soils. Associations of plants with endophytes can further enhance their phytoremediation potential. In this study, a rhizobox experiment was conducted to investigate whether inoculation with the root-colonizing fungus Piriformospora indica could further enhance the degradation of petroleum hydrocarbons in the root zone of maize (Zea mays L.). The rhizoboxes were subdivided into compartments in accordance with distance from the plants. After filling the boxes with soil from a petroleum-contaminated site, seedlings that had either been inoculated with P. indica or not were grown in the middle compartments of the rhizoboxes and grown for 64 days. A plant-free treatment was included for control. The presence of roots strongly increased the counts of total and petroleum-degrading soil bacteria, respiration, dehydrogenase activity, water-soluble phenols and petroleum degradation. All these effects were also found in the soil adjacent to the middle compartments of the rhizoboxes, but strongly decreased further away from it. Inoculation with P. indica further enhanced all the recorded parameters without changing the spatial pattern of the effects. Inoculated plants also produced around 40% more root and shoot biomass than noninoculated plants and had greener leaves. Together, the results indicate that the treatment effects on the recorded soil microbial and biochemical parameters including petroleum hydrocarbon degradation were primarily due to increased root exudation. Irrespectively of this, they show that maize can be used to accelerate the rhizodegradation of petroleum hydrocarbons in soil and that inoculation with P. indica can substantially enhance the phytoremediation performance of maize.     

Impact of Piriformospora indica on tomato growth and on interaction with fungal and viral pathogens

Piriformospora indica is a root endophytic fungus with plant-promoting properties in numerous plant species and induces resistance against root and shoot pathogens in barley, wheat, and Arabidopsis. A study over several years showed that the endophyte P. indica colonised the roots of the most consumed vegetable crop tomato. P. indica improved the growth of tomato resulting in increased biomass of leaves by up to 20%. Limitation of disease severity caused by Verticillium dahliae by more than 30% was observed on tomato plants colonised by the endophyte. Further experiments were carried out in hydroponic cultures which are commonly used for the indoor production of tomatoes in central Europe. After adaptation of inoculation techniques (inoculum density, plant stage), it was shown that P. indica influences the concentration of Pepino mosaic virus in tomato shoots. The outcome of the interaction seems to be affected by light intensity. Most importantly, the endophyte increases tomato fruit biomass in hydroponic culture concerning fresh weight (up to 100%) and dry matter content (up to 20%). Hence, P. indica represents a suitable growth promoting endophyte for tomato which can be applied in production systems of this important vegetable plant not only in soil, but also in hydroponic cultures.     

Piriformospora indica: a versatile root endophytic symbiont

Piriformospora indica, a unique root endophytic basidiomycete, exhibits symbiotic association with a variety of host plants. Spacio-temporally, the biphasic lifestyle of the fungus is characterized by distinct biotrophic and cell death associated root colonization processes. Fungal components such as fungal chitin induce specific host immune responses which also play an important role in establishing a successful sustainable interaction. The fungus-induced Pattern Triggered Immunity (PTI) is regulated by a complex interplay amongst the various phytohormones and anti-microbial molecules. The association so built up imparts a broad spectrum of benefits to the host viz., high salt tolerance, disease resistance, resistance to heavy metal toxicity, and growth-promotion involving value addition. These characteristics of P. indica can be translated into specific applications in agriculture and horticulture. The current article also outlines the future research prospects of this highly versatile fungus.     

Karyotype analysis,genome organization,and stable genetic transformation of the root colonizing fungus Piriformospora indica

Piriformospora indica (Basidiomycota, Sebacinales) is a root colonizing fungus which is able to increase biomass and yield of crop plants and to induce local and systemic resistance to fungal diseases and tolerance to abiotic stress. A prerequisite for the elucidation of the mode of action of this novel kind of symbiosis is knowledge of the genome organization as well as the development of tools to study and modify gene functions. Here we provide data on the karyotype and genetic transformation strategies. The fungus was shown to possess at least six chromosomes and a genome size of about 15.4–24 Mb. Sequences of the genes encoding the elongation factor 1-α (TEF) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used for genome size estimation through real-time PCR analysis. Chromosomal location investigated by Southern blot and expression analysis suggested that TEF and GAPDH are single-copy genes with strong and constitutive promoters. A genetic transformation system was established using a fragment of the TEF promoter region for construction of vectors carrying the selectable marker hygromycin B phosphotransferase. Results demonstrate that P. indica can be stably transformed by random genomic integration of foreign DNA and that it posses a relative small genome as compared to other members of the Basidiomycota.