Endophytic Relationship Research Articles

Endophytic entomopathogenic fungi as biological control agents of insect pests.

Entomopathogenic fungi capable of establishing mutualistic endophytic relationships with plants have a tremendous potential as biocontrol agents of insect pests. While fungi have long played an important and highly effective role in pest suppression, the utility of endophytic entomopathogenic fungi in pest management is a relatively new and emerging topic of biocontrol. Here we discuss the relevance of endophytic fungi to plant health in general, synthesize the current knowledge of the effectiveness of endophytic entomopathogenic fungi against diverse insect pests, discuss the indirect plant-mediated effects of endophytic entomopathogenic fungi on arthropods, and describe the diverse benefits of endophytic fungi to plants that are likely to affect herbivores and plant pathogens as well. Lastly, we consider major challenges to incorporating endophytic entomopathogenic fungi in biocontrol, such as their non-target effects and field efficacy, which can be variable and influenced by environmental factors. Continued research on endophyte-insect-plant-environment interactions is critical to advancing our knowledge of these fungi as a sustainable pest management tactic. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Seasonal Variation and Distribution Patterns of Endophytic Community in Withania somnifera

Withania somnifera also known as Ashwagandha used as rasayana (tonic) in Ayurvedic system of medicine. It is widely considered as Indian Ginseng and possesses medicinal properties like adaptogenic, antistress, anxiolytic, anti-arthritic, anti-inflammatory etc. There is long history of microbes having stable symbiotic relationship with plants aiding them in growth and survival. This endophytic relationship is also been observed in medicinal plants. This study is an attempt to observe the biodiversity of endophytic fungal community in Withania somnifera and to understand their colonization. The study was aimed to isolate endophytes from different organs of Withania somnifera with seasonal variations. A total of 131 endophytic fungi were isolated from 450 explants from different organs like leaves, stems and roots of W. somnifera. The overall colonization rate of endophytes in winter, summer and monsoon were 42.67%, 7.33% and 37.33% respectively. Among the identified isolates, most abundant and frequently isolated genera were Alternaria, Fusarium and Chaetomium sps. The dominant species isolated in winter were the Fusarium moniliformae and Chaetomium globosum from leaves with 17.24% and 13.79% dominant frequency, respectively. While Alternaria raphani and Chaetomium globosum were most dominant species in summer from leaves with 40% dominant density. In the monsoon, Microsporum ferruginem was the most dominant species isolated from leaves and stem with 61.29% and 42.86%, respectively. The most recurring endophytic species of Ascomycetes group were Alternaria alternata and Chaetomium globosum seen in all seasons.

Assessment of Phytochemicals, Antioxidant Activity and Enzyme Production of Endophytic Fungi Isolated from Medicinal Plant Sources

Endophytic fungi are mitosporic and meiosporic ascomycetes that asymptomatically reside in the internal tissues of plants beneath the epidermal cell layer, where fungi colonise healthy and living tissue via quiescent infections. Endophytes are important components of microbial diversity. Endophytic fungi isolated from medicinal plants more likely exhibit pharmaceutical potentials. These plentiful natural products isolated from endophytes represent a huge reservoir which offers an enormous potential for exploitation for medicinal, agricultural and industrial uses. There has been a great interest in endophytic fungi as potential producers of novel, biologically active products. Endophytes are believed to carry out a resistance mechanism to overcome pathogenic invasion by producing secondary metabolites. Globally, there are at least one million species of endophytic fungi in all plants which can potentially provide a variety of structurally unique natural products such as alkaloid, benzopyranones, chinones, flavanoids, phenols, steroids, xanthones and others. Therefore, there is an ample opportunity to unearth novel and interesting endophytic microorganisms with significant therapeutic efficacy. The objectives of our current study are to isolate endophytic fungi from specific medicinal plants found locally and carry out their characterization and isolation, followed by qualitative and quantitative assessment of secondary metabolites produced by them and study their antioxidant and enzyme activities. The present study, therefore would highlight the growing concept that the bioactive compounds produced by the endophytes not only establishes host endophyte relationship but also have an immense chance of application in the field of medicine, agriculture and industry.

Functional Importance of Endophytic Microorganisms in Plant Growth Promotion Bioactive Compound Production for Sustainable Agriculture

Endophytes constitute living microorganisms inhabiting inside tissues of plants. Endophytes perform criticalfunctions in upgrading the growth of plants and their defense to fight stress by a variety of phytohormones, biologically active compounds, volatile organic compounds, and biotechnologically valuable enzymes. How biological nitrogen fixation, nutrient uptake, and disease suppression occurs by endophytes, have been discussed in detail in the review. The mutual symbiotic relationship enhances plant growth, fitness, physiology, and metabolite production ability. Endophytes inhibit the invasion of pathogenic microorganisms and protect crops against diseases. Endophytes are also involved in strategies for environmental clean-up such as biodegradation, bioremediation, and phytoremediation. Therefore, it is necessary to analyze and study the mechanisms of interactions, colonisation, diversity, and functionalities for successful implications in agriculture. Thus, the endophytic relationship opens possibilities for medicine, agriculture, and biotechnology. The present review emphasizes the importance of endophytes in sustainable agriculture under several adverse environmental impacts through a better understanding of their functioning inside the plant.

Trichoderma Species: An Overview of Current Status and Potential Applications for Sustainable Agriculture

In agro-ecosystems, Trichoderma species are beneficial microorganisms that improve soil health and crop development. They form mutualistic endophytic relationships with a wide range of plant species, promoting host growth, protecting against pathogen attack, and improving micro-and macronutrient uptake and use efficiency. As a result, they should be promoted because they have the potential to improve agricultural sustainability while reducing the use of harmful chemicals in agriculture. This review provides an overview of the current and potential applications of Trichoderma species for sustainable agriculture, their beneficial roles and how they can be used to boost plant growth and crop yield.

Promotion of Arabidopsis immune responses by a rhizosphere fungus via supply of pipecolic acid to plants and selective augment of phytoalexins.

The ascomycete insect pathogenic fungi such as Metarhizium species have been demonstrated with the abilities to form the rhizosphere or endophytic relationships with different plants for nutrient exchanges. In this study, after the evident infeasibility of bacterial disease development in the boxed sterile soils, we established a hydroponic system for the gnotobiotic growth of Arabidopsis thaliana with the wild-type and transgenic strain of Metarhizium robertsii. The transgenic fungus could produce a high amount of pipecolic acid (PIP), a pivotal plant-immune-stimulating metabolite. Fungal inoculation experiments showed that M. robertsii could form a non-selective rhizosphere relationship with Arabidopsis. Similar to the PIP uptake by plants after exogenous application, PIP level increased in Col-0 and could be detected in the PIP-non-producing Arabidopsis mutant (ald1) after fungal inoculations, indicating that plants can absorb the PIP produced by fungi. The transgenic fungal strain had a better efficacy than the wild type to defend plants against the bacterial pathogen and aphid attacks. Contrary to ald1, fmo1 plants could not be boosted to resist bacterial infection after treatments. After fungal inoculations, the phytoalexins camalexin and aliphatic glucosinolate were selectively increased in Arabidopsis via both PIP-dependent and -independent ways. This study unveils the potential mechanism of the fungus-mediated beneficial promotion of plant immunity against biological stresses. The data also highlight the added values of M. robertsii to plants beyond the direct suppression of insect pest populations.

Gluconacetobacter diazotrophicus Gene Fitness during Diazotrophic Growth.

Plant growth-promoting (PGP) bacteria are important to the development of sustainable agricultural systems. PGP microbes that fix atmospheric nitrogen (diazotrophs) could minimize the application of industrially derived fertilizers and function as a biofertilizer. The bacterium Gluconacetobacter diazotrophicus is a nitrogen-fixing PGP microbe originally discovered in association with sugarcane plants, where it functions as an endophyte. It also forms endophyte associations with a range of other agriculturally relevant crop plants. G. diazotrophicus requires microaerobic conditions for diazotrophic growth. We generated a transposon library for G. diazotrophicus and cultured the library under various growth conditions and culture medium compositions to measure fitness defects associated with individual transposon inserts (transposon insertion sequencing [Tn-seq]). Using this library, we probed more than 3,200 genes and ascertained the importance of various genes for diazotrophic growth of this microaerobic endophyte. We also identified a set of essential genes. IMPORTANCE Our results demonstrate a succinct set of genes involved in diazotrophic growth for G. diazotrophicus, with a lower degree of redundancy than what is found in other model diazotrophs. The results will serve as a valuable resource for those interested in biological nitrogen fixation and will establish a baseline data set for plant free growth, which could complement future studies related to the endophyte relationship.

Establishment of Beauveria bassiana isolates as endophytes in rice cultivars and their biocontrol efficacy against rice stem borer, Sesamia calamistis

Possible endophytic colonisation of rice cultivar parts (leaves, stem and roots) by Beauveria bassiana isolates and their potential as biocontrol agents against Sesamia calamistis Hampson (African pink stem borer) were investigated. Five promising B. bassiana isolates were evaluated for their endophytic colonisation, the effectiveness of the inoculation methods and the efficacy of the isolates as biocontrol agents against S. calamistis. The plant part colonised is often dependent on the inoculation method. Colonisation of plant parts was assessed at 30 and 60 days after seed inoculation and foliar spray. For the pathogenicity activity, third instar larvae of S. calamistis were fed with rice stems that were previously inoculated with endophytic isolates of B. bassiana. Both inoculation methods led to the colonisation of the rice cultivar tissues, but were affected by the interactions of cultivars x isolates x inoculation methods. The colonisation of the cultivar plant parts varied over time (30- and 60-day intervals), and was affected by the inoculation method used. For both inoculation methods, highly significant differences were observed in the roots and the leaves over time (p=0.0001). However, with seed treatment, there was no significant difference in levels of colonisation in stems by the isolates x time (p=0.32). The B. bassiana isolates were pathogenic on the third instar larvae of S. calamistis, causing mortalities of more than 50% at 28 days after treatment. However, the virulence of the isolates varied. According to the isolates and the inoculation methods, B. bassiana formed an endophytic relationship with rice plants, and produced various mortality rates.
 Significance:
 
 Beauveria bassiana could be a potential biocontrol agent of rice stem borer, S. calamistis as there is no report of endophytic isolates of B. bassiana for the control of rice borers.
 Currently there is no commercially registered biocontrol agent against rice borers; hence further studies into B. bassiana could lead to the registration and commercialisation of B. bassiana as a bio-pesticide for rice stem borers.

Genome and secretome analysis of jute endophyte Grammothele lineata strain SDL-CO-2015-1: Insights into its lignocellulolytic structure and secondary metabolite profile

Grammothele lineata strain SDL-CO-2015-1, jute (Corchorus olitorius) endophyte has been reported to produce anti-cancer drug paclitaxel in culture condition. Here we investigated the genome using different bioinformatic tools to find its association with the production of commercially important compounds including taxol. Carbohydrate-active enzymes, proteases, and secretory proteins were annotated revealing a complex endophytic relationship with its plant host. The presences of a diverse range of CAZymes including numerous lignocellulolytic enzymes support its potentiality in biomass degradation. Genome annotation led to the identification of 28 clusters for secondary metabolite biosynthesis. Several biosynthesis gene clusters were identified for terpene biosynthesis from antiSMASH analysis but none could be specifically pinned to taxol synthesis. This study will direct us to understand the genomic organization of endophytic basidiomycetes with a potential for producing numerous commercially important enzymes and secondary metabolites taking G. lineata as a model.

Visualising confirmation of the endophytic relationship of Metarhizium anisopliae with maize roots using molecular tools and fluorescent labelling

ABSTRACTMetarhizium anisopliae, an entomopathogenic fungus, is found to contribute to plant nutrient uptake and condidered as a plant endophyte. However, there is very little visual evidence that demonstratrates the endophytic relationship. Since the incidence of Metarhizium within host plants may be ephemeral or of limited distribution within host tissue, visualisation is reliant on both presence and the ability to detect the fungus even when present at low levels. We inoculated maize seeds with an egfp-labelled isolate of M. anisopliae, and then detected the egfp fragment by qualitative and quantitative PCR, and examined fluorescence of mycelia in the inoculated roots by laser scanning confocal microscopy (LSCM). The optimised PCR qualitatively identified the presence of egfp-labelled M. anisopliae at a 1:3000 M. anisopliae-maize DNA ratio. Root samplings of inoculated plants at intervals over 24 days were discovered egfp-labelled fragments with 14-d roots having the highest proportion with amplification. The egfp-label concentrations ranged very low in 2.976 × 10−7.42∼−7.02 ng/μL, with a rise process in 4–11 days after M. anisopliae treatment. Under LSCM, fluorescent mycelia in roots were identified, and the clear images were captured to show hyphal development extending along a longitudinal line of root cells and even forming a hyphal mass in the intercellular space. There were many hyphae at the branch junctions of the lateral roots. The methodology used to detect the fungus will be important for exploring the multi-trophic function of M. anisopliae and co-evolution in plants.

Beneficial effects of endophytic fungi colonization on plants.

Due to increasingly limited water resources, diminishing farmland acreage, and potentially negative effects of climate change, an urgent need exists to improve agricultural productivity to feed the ever-growing population. Plants interact with microorganisms at all trophic levels, adapting growth, developmental, and defense responses within a complicated network of community members. Endophytic fungi have been widely reported for their ability to aid in the defense of their host plants. Currently, many reports focus on the application of endophytic fungi with the capability to produce valuable bioactive molecules, while others focus on endophytic fungi as biocontrol agents. Plant responses upon endophytic fungi colonization are also good for the immune system of the plant. In this paper, the possible mechanisms between endophytic fungi and their hosts were reviewed. During long-term evolution, plants have acquired numerous beneficial strategies in response to endophytic fungi colonization. The interaction of endophytic fungi with plants modulates the relationship between plants and both biotic and abiotic stresses. It has previously been reported that this endophytic relationship confers additional defensive mechanisms on the modulation of the plant immune system, as the result of the manipulation of direct antimicrobial metabolites such as alkaloids to indirect phytohormones, jasmonic acid, or salicylic acid. Furthermore, plants have evolved to cope with combinations of stresses and experiments are required to address specific questions related to these multiple stresses. This review summarizes our current understanding of the intrinsic mechanism to better utilize these benefits for plant growth and disease resistance. It contributes new ideas to increase plant fitness and crop productivity.

The puzzle of highly virulent Metarhizium anisopliae strains from Annona squamosa fields against Helicoverpa armigera.

In our search for indigenous virulent strains of the entomopathogenic fungi, we observed that Metarhizium isolates from soils associated with Annona squamosa (custard apple) have higher virulence (>90% mortality of Helicoverpa armigera larvae at 1/10th spore concentration) than strains isolated from Solanum lycopersicum (tomato) fields. Proteomic analysis revealed two insecticidal cyclopeptides of A. squamosa origin in the M. anisopliae strains that led to higher virulence against H. armigera. Transcriptomic and genomic data indicated that M. anisopliae strains and A. squamosa had more than 20 genes in common, including those for cyclic hexapeptide synthase, non-ribosomal peptide synthetase, and plant cyclotide genes, which are involved in the biosynthesis of insecticidal cyclopeptides. These genes were absent in M. anisopliae strains isolated from the S. lycopersicum fields. Further, these strains can establish an endophytic relationship with A. squamosa suggesting that these rhizospheric strains originally could be endophytes, which were eventually released into the soil. Further, Metarhizium strains associated with Capsicum annuum (chili), Azadirachta indica (neem), and Carica papaya (papaya) - plants with insecticidal properties - also had higher virulence against H. armigera. Thus exploration of rhizospheres of plants producing insecticidal metabolites to isolate entomopathogenic fungi, per se, could be a viable strategy in agricultural for crop protection.

Plant trichomes as microbial habitats and infection sites

Trichomes, also simply referred to as hairs, are fine outgrowths of epidermal cells in many organisms including plants and bacteria. Plant trichomes have long been known for their multiple beneficial roles, ranging from protection against insect herbivores and ultraviolet light to the reduction of transpiration. However, there is increasing evidence that the presence of trichomes may have detrimental consequences for plants. For example, plant pathogenic bacteria can enter hosts through the open bases or broken stalks of damaged trichomes. Similarly, trichomes are considered a preferred site for fungal infection, and in this regard, the colonization and penetration of trichomes by fungi and oomycetes have been visualized using light, fluorescence, and scanning electron microscopy in a variety of plants from grasses to shrubs and trees. In addition to parasitic interactions, trichomes also form a host site for endophytic relationships with fungi, thereby serving as an unusual fungal niche. The replication and presence of plant viruses in trichomes have also been confirmed after inoculation. In contrast, the well-known beneficial Azolla–Anabaena symbiosis is facilitated through epidermal trichomes of the seedless vascular plant Azolla. These observations indicate that plant trichomes are involved in multiple interactions in terms of providing microbial habitats and infection sites as well as functioning as protective structures. Trichome-related microbial parasitism and endophytism can, in many ways, be considered comparable to those associated with root hairs.

ASSESSMENT OF SECONDARY METABOLITES WITH RELATION TO THEIR ANTIOXIDANT ACTIVITY OF FUNGAL ENDOPHYTES ISOLATED FROM MEDICINAL PLANTS

Objective: The detection of fungal endophytes and assessment of their antioxidant activity isolated from two medicinally important plants, Calotropis procera (L.) R. Br. and Catharanthus roseus (L.) G. Don. from parts of Burdwan district of West Bengal, India.Methods: Isolation of fungal endophytes was carried out following the standard methods in potato dextrose agar (PDA) media. Identification, determination of colonization frequency, fermentation and extraction was done as stated in the standard protocol. Qualitative assay of the bioactive compound has investigated by standard protocol specifically for them. Quantitative estimation of total phenol was done by spectrophotometric method using gallic acid as standard. The antioxidant activity of the cell-free extract was determined by phosphomolybdenum assay and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging method.Results: Three dominant endophytic fungi each from C. procera and C. roseus with high colonization frequency in Penicillium singorense 25% and Alternaria alternata 21.87 % respectively has isolated and identified. Qualitative detection of secondary metabolites revealed that Penicillium singorense isolated from both plants and Aspergillus neoflaviceps (from C. procera) were found to be able to produce all the functional metabolites so far tested. It is evident that highest phenol 27.65±1.2 mg/g and flavonoid 2.06±0.1 mg/g content was shown by Curvularia geniculata. Highest antioxidant activity was exhibited by C. geniculata in both phosphomolybdenum assay i.e., total antioxidant capacity 2.46±0.11 and % inhibition of DPPH radical 94.55±0.015%.Conclusion: Present study, therefore highlights the growing concept that the bioactive compounds produced by the endophytes not only established host endophyte relationship but also have an immense chance of an application in the field of medicine, agriculture and industry.

Fusarium culmorum isolated from rhizosphere of wooly cupgrass (Eriochloa villosa) in Debrecen (East Hungary)

Wooly cupgrass (Eriochloa villosa) is an East-Asian originated weed species and it has been spreaded worldwide by now. The first occurrence of this species in Hungary was observed and published in 2008 nearby Gesztely village (Borsod-Abaúj-Zemplén county, North-East Hungary) than in the summer of 2011 a significant population was discovered next to Debrecen city (Hajdú-Bihar county, East Hungary).
 In 2013 this weed was also reported from Szentborbás village, Somogy county (South-West Hungary). These observations of spreading and its biological features (production of stolons and large number of seeds, moreover herbicide tolerance) indicate that wooly cupgrass (E. villosa) has a great potential of invasiveness, so it may become a hazardous weed not only in Hungary but in all over the world.
 The objective of this study was to identify the fungus which was isolated from wooly cupgrass (E. villosa) root residue samples which were collected after maize harvesting on arable land in late autumn, near Debrecen. The identification of the fungus based on morphological characters of colonies and the features of conidia developed on potato dextrose agar (PDA) plates. After the examination of axenic culture we revealed that the fungus from rhizosphere of wooly cupgrass was Fusarium culmorum. Pathogenicity and/or endophytic relationship between the fungus and wooly cupgrass is still uncertain so pathogenicity tests and reisolations from plants are in progress.

Endophytic Association of Trichoderma asperellum within Theobroma cacao Suppresses Vascular Streak Dieback Incidence and Promotes Side Graft Growth

Trichoderma species are able to persist on living sapwood and leaves of cacao (Theobroma cacao) in an endophytic relationship. In this research, we evaluated the ability of Trichodema asperellum introduced at the incision site in the bark for side grafting with the concentration of 4 g/10 mL, 4 g/100 mL, and 4 g/1,000 mL (suspended in water) in suppressing vascular streak dieback (VSD) incidence and promoting growth of side grafts in the field. The incidence of VSD in two local clones of cacao, MCC1 and M04, without application of T. asperellum was 71.2% and 70.1% at 21 wk after grafting, respectively. However, when the two clones were treated with a concentration of 4 g/10 mL T. asperellum, the incidence was 20.6% and 21.7%, respectively, compared to 29.1% and 20.9% at 4 g/100 mL and 18.2% and 15.6% at 4 g/1,000 mL. By comparing to the control, the treatment with the same concentrations of T. asperellum listed above, the total number of stomata in MCC1 decreased by 41.9%, 30.2%, and 14.0% and in M04 by 30.5%, 21.9%, and −2.5% (exception), respectively. Otherwise, the total area of stomata opening increased by 91.4%, 99.7%, and 28.6% in MCC1 and by 203.8%, 253.5%, and 35.9% in M04, respectively. Furthermore, the number of buds and branches treated with a mixture concentration on the the two clones increased by 90.7% and 21.7%, respectively. These data showed that the application of T. asperellum to cacao scions while grafting can decrease VSD incidence in side grafts and increase growth of grafts in addition to decreasing total number of stomata, increasing total area of opened stomata, and increasing number of buds and branches.

Hidden Host Plant Associations of Soilborne Fungal Pathogens: An Ecological Perspective

Much of the current knowledge on population biology and ecology of soilborne fungal pathogens has been derived from research based on populations recovered from plants displaying disease symptoms or soil associated with symptomatic plants. Many soilborne fungal pathogens are known to cause disease on a large number of crop plants, including a variety of important agronomical, horticultural, ornamental, and forest plants species. For instance, the fungus Verticillium dahliae causes disease on >400 host plants. From a phytopathological perspective, plants on which disease symptoms have not been yet observed are considered to be nonhosts for V. dahliae. This term may be misleading because it does not provide information regarding the nature of the plant-fungus association; that is, a nonhost plant may harbor the fungus as an endophyte. Yet, there are numerous instances in the literature where V. dahliae has been isolated from asymptomatic plants; thus, these plants should be considered hosts. In this article, we synthesize scattered research that indicates that V. dahliae, aside from being a successful and significant vascular plant pathogen, may have a cryptic biology on numerous asymptomatic plants as an endophyte. Thus, we suggest here that these endophytic associations among V. dahliae and asymptomatic plants are not unusual relationships in nature. We propose to embrace the broader ecology of many fungi by differentiating between "symptomatic hosts" as those plants in which the infection and colonization by a fungus results in disease, and "asymptomatic hosts" as those plants that harbor the fungus endophytically and are different than true nonhosts that should be used for plant species that do not interact with the given fungus. In fact, if we broaden our definition of "host plant" to include asymptomatic plants that harbor the fungus as an endophyte, it is likely that the host ranges for some soilborne fungal pathogens are much larger than previously envisioned. By ignoring the potential for soilborne fungal pathogens to display endophytic relationships, we leave gaps in our knowledge about the population biology and ecology, persistence, and spread of these fungi in agroecosystems.

Incidence of ‘Candidatus Liberibacter europaeus’ and phytoplasmas in Cacopsylla species (Hemiptera: Psyllidae) and their host/shelter plants

Psyllids, as vectors of phloem-restricted plant pathogens, are serious agricultural pests. Fruit tree phytoplasmas are transmitted by different Cacopsylla spp., while other psyllids are known vectors of liberibacters. Recently, the bacterium ‘Candidatus Liberibacter europaeus’ was found in pear trees and in Cacopsylla pyri (Linnaeus), the vector of ‘Ca. Phytoplasma pyri’. This new species does not cause symptoms in plants and is probably a symbiont rather than a pathogen. Based on these findings and the assumption that ‘Ca. Liberibacter europaeus’ is widespread, we studied its distribution in the genus Cacopsylla and in the respective host and shelter plants (where psyllids aestivate and overwinter), as well as its possible co-presence with ‘Ca. Phytoplasma’ spp. We tested 14 Cacopsylla species and 11 plant species from northwestern Italy, Hungary and Israel, characterized by warm oceanic, temperate continental and warm Mediterranean climatic conditions, respectively. ‘Ca. Liberibacter europaeus’ was common within the Cacopsylla genus, being present in nine of the 14 species screened as well as in most host plants, whereas none of the shelter plants tested positive for this bacterium. Altogether, these findings indicate the presence of ‘Ca. Liberibacter europaeus’ in continental zones, whereas it does not seem to be widespread in the Mediterranean region. Furthermore, lack of specific symptoms in all infected plants confirms an endophytic relationship with this bacterium, while its abundance in insects suggests a beneficial role for the host. Co-infections with phytoplasmas, observed in insects and plants, require further study to evaluate the possible interactions between them.

A fungal endophyte reinforces population adaptive differentiation in its host grass species

Hereditary symbioses between fungal endophytes and grasses are relatively recent in the history of plant life. Given < 80 million yr of co-evolution, symbioses are likely to have impacted plant microevolutionary rather than macroevolutionary processes. Therefore, we investigated the microevolutionary role of the fungal endophyte Neotyphodium lolii in the adaptive differentiation of its host species Lolium perenne. Endophyte frequency in 22 natural L. perenne populations was established across a water availability gradient. Adaptive differentiation among five populations, and between symbiotic (S) and nonsymbiotic (NS) plants, was examined in a glasshouse experiment under nonlimiting and limiting water conditions. Genetic differentiation was subsequently assessed among populations, and between S and NS individuals, using 14 simple sequence repeats (SSR). Symbiosis frequencies were positively correlated to water availability. Adaptive population differentiation occurred following a trade-off between biomass production under nonlimiting water conditions and survivorship under water stress. Endophytic symbiosis increased plant survival in xeric populations, and reinforced competitiveness in mesic populations. No genetic difference was detected between S and NS plants within populations. Therefore, we conclude that the endophyte relationship is responsible for these effects. Local adaptation of the host plant, appears to be supported by the fungal endophyte.

Fungi associated with the southern Eurasian orchid Spiranthes spiralis (L.) Chevall

The hitherto unknown relationships between the European orchid Spiranthes spiralis (L.) Chevall and its internally associated fungi were explored by a combined approach involving microscopy-based investigations at a morpho-histological level as well as by molecular analyses of the identity of the eukaryotic endophytes present in the root tissue of the plant. We found that this orchid which is currently reported to have a vulnerable status in northern Italy, can host and interact with at least nine types of fungi. Some of these fungi show similarity to mycorrhizal genera found in orchids such as the Ceratobasidium-Rhizoctonia group. Other fungi found are from the genera Davidiella (Ascomycota), Leptosphaeria (Ascomycota), Alternaria (Ascomycota), and Malassezia (Basidiomycota), some of which until have not previously been reported to have an endophytic relationship with plants. The repeated occurrence of often pathogenic fungi such as Fusarium oxysporum, Bionectria ochroleuca, and Alternaria sp., within healthy specimens of this orchid suggests a tempered interaction with species that are sometimes deleterious to non-orchid plants. The fact is reminiscent of the symbiotic compromise established by orchids with fungi of the rhizoctonia group.