Endophytes In Seeds Research Articles

Xanthomonas sontii, and Not X. sacchari, Is the Predominant Vertically Transmitted Core Rice Seed Endophyte.

Seed endophytes, particularly the abundant, core, and vertically transmitted species, are major areas of focus in host microbiome studies. Apart from being the first members to colonize, they accompany the plant throughout its development stages and to the next generation. Recently published studies have reported the keystone species to be Xanthomonas sacchari, a core endophyte that is vertically transmitted in rice with probiotic properties. Furthermore, the Xanthomonas species was reported to be involved in the assembly of beneficial bacteria after early inoculation in rice seeds. However, the strains discussed in these studies were misclassified as X. sacchari, a well-known pathogen of sugarcane. By including nonpathogenic Xanthomonas species with plant-protective functions reported from rice seeds, we have correctly established the phylogenetic and taxonomic identity of the keystone species as X. sontii. This will enable researchers to use the correct reference or lab strain of X. sontii for further systematic and in-depth studies as a model endophyte in plant-microbe interactions apart from its exploitation in seed health.

Structure and Diversity of Endophytic Bacteria in Maize Seeds and Germinating Roots.

Seed endophytes in maize, which facilitate the transmission of microorganisms from one plant generation to the next, may play a crucial role in plant protection and growth promotion. This study aimed to investigate the effects of various maize varieties on the communities of endophytic bacteria in seeds and germinating roots. This study utilized Illumina high-throughput sequencing technology to examine the structural and diversity differences of endophytic bacterial communities within seed maize (BY1507), silage maize (QQ446), and wild maize (Teosinte) in both seeds and germinating roots. The results showed that 416 bacterial genera were detected, with Pantoea, Lachnospiraceae, Pararhizobium, Enterobacteriaceae, Stenotrophomonas, and Pseudonocardia being the most prevalent (relative abundance > 10%) at the genus level. No significant difference was observed in diversity indices (Chao1, ACE, Shannon, and Simpson) of seed endophytes among BY1507, QQ446, and Teosinte. The Shannon and Simpson indices for the germinating root endophyte from the wild variety (Teosinte) were significantly higher than the domesticated varieties (BY1507 and QQ446). PCoA revealed a notable overlap in the endophytic bacterial communities from the seeds of BY1507, QQ446, and Teosinte. Yet, clustering patterns were found. Co-occurrence network analysis showed that BY1507, QQ446, and Teosinte share a notable proportion of shared endophytic bacteria (>30%) between the seeds and germinating roots. This investigation elucidates the characteristics of endophytic microbial communities of seeds and germinating roots with seed maize, silage maize, and wild maize, offering data for future research on the physiological ecological adaptation of these endophytic microbial communities.

Seed endophytes reshape rhizosphere microbiota to promote the growth of Eucommia ulmoides seedlings

Plant seeds harbor numerous beneficial endophytes that can be vertically transmitted across generations, positively influencing plant growth. This study aimed to enhance our understanding of the functions of endophytes in Eucommia ulmoides seeds and investigate their potential mechanisms for promoting E. ulmoides seedling growth. Preliminary screening for seed endophytes that increase E. ulmoides seedling growth was conducted using microbe–seedling co-culture experiments. Subsequently, the two strains with the most pronounced growth-promoting effects were tested in pot experiments. The mechanism underlying their effects on E. ulmoides growth was elucidated through high-throughput sequencing analysis of rhizosphere soil. The bacterium Cytobacillus gottheilii B7 and the fungus Cladosporium halotolerans Z27 had the most significant growth-promoting effects on E. ulmoides seedlings. Inoculation of these strains into the soil led to substantial improvements in physiological indicators in E. ulmoides. Notably, strain B7 showed a superior effect compared to strain Z27. Although strain B7 inoculation reduced rhizosphere bacterial α-diversity, it strengthened the network interactions among rhizosphere microorganisms and selectively recruited specific rhizosphere microbial taxa, including Ilumatobacter, Algoriphagus and OLB13, while inhibiting the growth of Subulicystidium and Symmetrospora. Functional annotation suggested that strain B7 inoculation enhanced plant nitrite respiratory metabolism and inhibited plant pathogen. Similarly, strain Z27 inoculation recruited genera such as Flavihumibacter, Pseudoxanthomonas, and Myriococcum, while inhibiting Symmetrospora. These results provide scientific data for a comprehensive analysis of seed endophytes in the future and valuable resources for the development and application of microbial inoculants.

Recent progress in the role of seed endophytic bacteria as plant growth-promoting microorganisms and biocontrol agents.

The importance of microorganisms residing within the host plant for their growth and health is increasingly acknowledged, yet the significance of microbes associated with seeds, particularly seed endophytic bacteria, remains underestimated. Seeds harbor a wide range of bacteria that can boost the growth and resilience of their host plants against environmental challenges. These endophytic associations also offer advantages for germination and seedling establishment, as seed endophytic bacteria are present during the initial stages of plant growth and development. Furthermore, plants can selectively choose bacteria possessing beneficial traits, which are subsequently transmitted through seeds to confer benefits to future generations. Interestingly, even with the ongoing discovery of endophytes in seeds through high-throughput sequencing methods, certain endophytes remain challenging to isolate and culture from seeds, despite their high abundance. These challenges pose difficulties in studying seed endophytes, making many of their effects on plants unclear. In this article, a framework for understanding the assembly and function of seed endophytes, including their sources and colonization processes was outlined in detail and available research on bacterial endophytes discovered within the seeds of various plant species has also been explored. Thus, this current review aims to provide valuable insights into the mechanism of underlying seed endophytic bacteria-host plant interactions and offers significant recommendations for utilizing the seed endophytic bacteria in sustainable agriculture as plant growth promoters and enhancers of environmental stress tolerance.

Diversity and functional traits of seed endophytes of Dysphania ambrosioides from heavy metal contaminated and non-contaminated areas.

Seed endophytes played a crucial role on host plants stress tolerance and heavy metal (HM) accumulation. Dysphania ambrosioides is a hyperaccumulator and showed strong tolerance and extraordinary accumulation capacities of multiple HMs. However, little is known about its seed endophytes response to field HM-contamination, and its role on host plants HM tolerance and accumulation. In this study, the seed endophytic community of D. ambrosioides from HM-contaminated area (H) and non-contaminated area (N) were investigated by both culture-dependent and independent methods. Moreover, Cd tolerance and the plant growth promoting (PGP) traits of dominant endophytes from site H and N were evaluated. The results showed that in both studies, HM-contamination reduced the diversity and richness of endophytic community and changed the most dominant endophyte, but increased resistant species abundance. By functional trait assessments, a great number of dominant endophytes displayed multiple PGP traits and Cd tolerance. Interestingly, soil HM-contamination significantly increased the percentage of Cd tolerance isolates of Agrobacterium and Epicoccum, but significantly decreased the ration of Agrobacterium with the siderophore production ability. However, the other PGP traits of isolates from site H and N showed no significant difference. Therefore, it was suggested that D. ambrosioides might improve its HM tolerance and accumulation through harboring more HM-resistant endophytes rather than PGP endophytes, but to prove this, more work need to be conducted in the future.

Germination of pecan seeds changes the microbial community.

Endophytes are core of the plant-associated microbiome, and seed endophytes are closely related to the plant growth and development. Seed germination is an important part of pecan's life activities, but the composition and changes of microbes during different germination processes have not yet been revealed in pecan seeds. In order to deeply explore the characteristics of endophytes during the germination process of pecan, high-throughput sequencing was performed on seeds at four different germination stages. Findings of present study was found that the diversity and composition of microorganisms were different in different germination stages, and the microbial richness and diversity were highest in the seed endocarp break stage. It was speculated that the change of endophytes in pecan seeds was related to the germination stage. By evaluating the relationship between microbial communities, the core microbiota Cyanobacteria, Proteobacteria and Actinobacteria (bacterial) and Anthophyta and Ascomycota (fungal) core microbiota were identified in germinating pecan seeds. Finally, biomarkers in different germination processes of pecan seeds were identified by LEfSe analysis, among which Proteobacteria, Gamma proteobacteria and, Cyanobacteria and Ascomycota and Sordariomycetes were most abundant. Thus, this study will help to explore the interaction mechanism between pecan seeds and endophytes in different germination processes, and provide materials for the research and development of pecan seed endophytes.

Community Structure and Diversity of Endophytic Bacteria in Melon (Cucumis melo L.) Seeds

Endophytic bacteria play a crucial role in the life cycle of Spermatophyta, and exploring the beneficial interactions between seeds and endophytic bacteria is an effective strategy for sustainable agricultural development and improved crop productivity. In this study, we employed Illumina Hiseq high-throughput sequencing technology to analyze the amplified fragments of microbial 16S rRNA sequences from melon seeds of MR-1, HH and their F1 generation, which displayed varying levels of resistance to bacterial fruit blotch disease. Initially, we extracted endophytes from eight melon samples and performed sequencing, obtaining an average of 84,758 effective tags per sample. At the 97% similarity level, the number of operational taxonomic units (OTUs) derived from melon seed endophytes ranged from 447 to 492. After filtering out low-level OTUs, we obtained an average of 65,866 valid tags from the eight samples, representing approximately 160 genera and 167 species across eight phyla. It was found that the composition of the endophyte communities in the eight samples was basically similar, and their abundance showed some variability. Furthermore, we utilized the KO and COG databases to functionally predict the genes of endophytes in melon seeds. The analysis revealed a significant enrichment of genes associated with pathways related to protein synthesis, carbohydrate metabolism, substance transport, and metabolite synthesis. These findings greatly contribute to our understanding of endophytic bacteria in melon seeds and serve as a vital reference for the selection and breeding of high-quality melon seeds, as well as the prevention of seed diseases.

Plant Protective and Growth Promoting Effects of Seed Endophytes in Soybean Plants.

Seed-borne diseases reduce not only the seed germination and seedling growth but also seed quality, resulting in the significant yield loss in crop production. Plant seed harbors diverse microbes termed endophytes other than pathogens inside it. However, their roles and application to agricultures were rarely understood and explored to date. Recently, we had isolated from soybean seeds culturable endophytes exhibiting in-vitro antagonistic activities against common bacterial and fungal seed-borne pathogens. In this study, we evaluated effects of seed treatment with endophytes on plant growth and protection against the common seed-borne pathogens: four fungal pathogens (Cercospora sojina, C. kikuchii, Septoria glycines, Diaporthe eres) and two bacterial pathogens (Xanthomonas axonopodis pv. glycines, Pseudomonas syringae pv. tabaci). Our experiments showed that treatment of soybean seeds with seed endophytes clearly offer protection against seed-borne pathogens. We also found that some of the endophytes promote plant growth in addition to the disease suppression. Taken together, our results demonstrate agricultural potential of seed endophytes in crop protection.

Endophytic Seed-Associated Bacteria as Plant Growth Promoters of Cuban Rice (Oryza sativa L.).

Cuban rice cultivars INCA LP-5 and INCA LP-7 are widely distributed in Cuba and Caribbean countries. Although there are studies about rhizospheric bacteria associated with these cultivars, there are no reports about their seed-associated bacteria. This study aimed to isolate endophytic bacteria from rice seeds and select those with the greatest plant growth-promoting traits. A total of nineteen bacterial strains from the genera Pantoea, Bacillus, Paenibacillus, and Pseudomonas were isolated from the husk and endosperm of rice seeds. The strains Pantoea sp. S5-1, Pseudomonas sp. S5-38, and Pseudomonas sp. S7-1 were classified as the most promissory to increase rice growth as they demonstrated the presence of multiple plant growth-promoting traits such as the production of auxins, phosphate, and potassium solubilization, the production of siderophores, and the inhibition of the phytopathogen Pyricularia oryzae. The inoculation of strains of Pantoea sp. and Pseudomonas spp. in rice improves the height, root length, fresh weight, and dry weight of the shoot and root after 21 days post-inoculation in hydroponic assays. This study constitutes the first report on Cuban rice cultivars about the presence of endophytes in seeds and their potential to promote seedling growth. Pantoea sp. S5-1, Pseudomonas sp. S5-38, and Pseudomonas sp. S7-1 were selected as the more promising strains for the development of bio-stimulators or bio-inoculants for Cuban rice crops.

Assembly and Function of Seed Endophytes in Response to Environmental Stress.

Seeds are colonized by diverse microorganisms that can improve the growth and stress resistance of host plants. Although understanding the mechanisms of plant endophyte-host plant interactions is increasing, much of this knowledge does not come from seed endophytes, particularly under environmental stress that the plant host grows to face, including biotic (e.g., pathogens, herbivores and insects) and abiotic factors (e.g., drought, heavy metals and salt). In this article, we first provided a framework for the assembly and function of seed endophytes and discussed the sources and assembly process of seed endophytes. Following that, we reviewed the impact of environmental factors on the assembly of seed endophytes. Lastly, we explored recent advances in the growth promotion and stress resistance enhancement of plants, functioning by seed endophytes under various biotic and abiotic stressors.

Characterization and diversity of seed endophytic bacteria of the endemic holoparasitic plant Cistanche armena (Orobanchaceae) from a semi-desert area in Armenia

AbstractWe explored the seed-associated bacterial endophytic microbiome in seeds of the endemic holoparasitic species Cistanche armena from a saline and arid habitat in Armenia. A combination of culture-dependent and molecular techniques was employed for identifying the seed endomicrobiome (culturable and unculturable). From surface-sterilized seeds, 10 phyla, comprising 256 endophytic bacterial genera, were identified. Of the culturable strains, we also investigated the plant growth-promoting (PGP) traits. Most of the isolates were spore forming, halotolerant and alkaliphile Bacillus spp., indicating that the endophytic bacteria of C. armena seeds own traits related to the natural habitat of their host plant. Our results confirm that Bacillus species are common and dominated endophytes from plants growing on saline and arid soils. Pantoea spp. and Stenotrophomonas spp. are more favourable PGP endophytes in seeds of C. armena. The PGP traits of these bacteria, such as production of indole, a precursor of auxin, ACC-deaminase and organic acids have the potential to improve the tolerance of their host plants against the abiotic stresses present in their natural habitat. To the best of our knowledge, this is the first report concerning bacterial seed endophytes of the C. armena.

Culture dependent and independent characterization of endophytic bacteria in the seeds of highland barley.

Endophytes in the seeds of plants have shown plant growth promoting (PGP) properties. Highland barley is an economically important crop and a major part of the local diet in the Tibetan Plateau, China, with potential health benefits. We applied culture-dependent and culture-independent methods to study endophytic bacteria in the seeds of eight Highland barley varieties. Based on the seed properties, the variety Ali was clearly separated from the other varieties except the variety CM. Most of the 86 isolates were assigned into genus Bacillus. Approximately half of the isolates showed PGP properties in vitro. Compared to the not-inoculated plants, inoculation with the isolate Bacillus tequilensis LZ-9 resulted in greater length and number of roots, and in bigger aboveground and root weights. Based on the 16S rRNA gene sequencing, the seed microbiome was majorly affiliated with the phylum Proteobacteria and the family Enterobacteriaceae. Overall, the bacterial community compositions in the different varieties were different from each other, yet the between variety differences in community composition seemed relatively small. The differences in community compositions were associated with differences in the total and reducing sugar contents and viscosity of the seeds, thus possibly connected to differences in the osmotic pressure tolerance of the endophytes. The results suggested that the seed endophytes are likely to promote the growth of Highland barley since germination.

Temporal Dynamics of Endogenous Bacterial Composition in Rice Seeds During Maturation and Storage, and Spatial Dynamics of the Bacteria During Seedling Growth.

Seed endophytes are of interest because they are believed to affect seed quality, and ultimately, plant growth and fitness. A comprehensive understanding of the assembly of the seed microbiome during seed development and maturation, the fate of microbes during storage, and the migration of microbes during seedling growth are still lacking. In this study, to understand the assembly and fate of endogenous bacteria in rice seeds from the ripening stage to the storage and seedling stages, we employed culture-dependent and metagenomic analyses. Bacterial communities in rice seeds were composed of a few dominant taxa that were introduced at the milky and dough stages, and they persisted during seed maturation. The culturable bacterial population gradually increased during the ripening stage, whereas there was a gradual decrease during storage. Bacteria that persisted during storage proliferated after imbibition and were distributed and established in the shoots and roots of rice seedlings. The storage temperature influenced the abundance of bacteria, which consequently changed the bacterial composition in the shoots and roots of seedlings. Pantoea, Pseudomonas, and Allorhizobium were consistently abundant from seed development to the germination stage. Some endogenous bacterial strains significantly promoted the growth of Arabidopsis and rice plants. Overall, our results indicate that rice seeds are colonized by a few bacterial taxa during seed development, and their relative abundance fluctuates during storage and contributes significantly to the establishment of endophytes in the stems and roots of rice plants. The selected bacterial isolates can be used to improve the growth and health of rice plants. To the best of our knowledge, this is the first study to reveal the dynamics of bacterial populations during storage of rice seeds at different temperatures. The temporal dynamics of the bacterial community during seed storage provide clues for the manipulation of endogenous bacteria in rice plants.

Inoculated Seed Endophytes Modify the Poplar Responses to Trace Elements in Polluted Soil

Seed endophyte inoculation can enhance the plant tolerance to pollutants, which allows plant cultivation on trace element (TE) polluted soils. Methylobacterium sp. CP3 and Kineococcus endophyticus CP19 were tested in vitro for their tolerance to Zn and Cd and their plant growth promotion traits. The in vivo effects of bioaugmentation with individual strains or both strains were tested using two poplar cultivars, Populus deltoides x (P. trichocarpa x P. maximowiczii) ‘Dender’ and ‘Marke’, grown in TE polluted soil for six weeks. Methylobacterium sp. was found to grow on media enriched with 0.4 and 0.8 mM Cd, and both endophytes tolerated 0.6 and 1 mM Zn, due to the presence of genes involved in Zn and Cd tolerance and transport. Methylobacterium sp. showed an extracellular ion sequestration mechanism. Production of indole-3-acetic acid by Methylobacterium sp. and K. endophyticus, as well as phosphorus solubilization by Methylobacterium sp. were observed. Bioaugmentation with both endophytes increased the shoot length of Populus ‘Marke’ and enhanced the Mg uptake in both cultivars. Inoculation with Methylobacterium sp. reduced the bioaccumulation of Zn in ‘Marke’, conferring it an excluder strategy. Methylobacterium sp. and K. endophyticus seemed to improve the plant nutritional status, which can alleviate abiotic stress.

Drivers of richness and community composition of fungal endophytes of tree seeds.

Recent studies revealed a high diversity of fungal endophytes in traded tree seeds, including potential plant pathogens. The factors determining richness and composition of seed mycobiomes are poorly understood, but might be an important determinant for tree health. We assessed the relative impact of host identity, site, several site-specific environmental factors, and whether the host was sampled in its native or non-native distribution range, on the richness and composition of fungal seed endophytes of nine tree species across 15 sites in Europe and North America. Our results show that fungal richness was affected by host identity, but not by environmental variables or host distribution range. Fungal community composition was primarily driven by host identity, and to a lesser extent by environment. Around 25% of the 2147 amplicon sequence variants (ASVs) were generalists appearing on both continents and in both gymnosperms and angiosperms. Around 63% of the ASVs appeared in only gymnosperms or angiosperms, and 33% of the ASVs were associated with a single host species, while none were found in all tree species. Our results suggest that although seed trade might facilitate movements of fungi, their establishment and spread in the new environment might be limited by host availability.

Harnessing chickpea (Cicer arietinum L.) seed endophytes for enhancing plant growth attributes and bio-controlling against Fusarium sp.

A seed microbiome is likely to have important impacts on plant fitness and productivity but functional potentials of seed microbiome remain poorly understood. It is also suggested that bio-inoculants developed from or compatible with seed microbiome are more likely to produce desired outcomes of sustainable increase in agriculture productivity but few empirical evidences are available. The aim of this study was to identify culturable endophytes of the germinating and dry seeds of chickpea (Cicer arietinum L.), and their functional attributes. We isolated 29 bacterial strains from chickpea seeds (8 strains from dry and 21 strains from germinating seeds). Phylogenetic analysis based on 16S rDNA showed that the seed endophytic bacteria belong to Enterobacter sp., Bacillus sp., Pseudomonas sp., Staphylococcus sp., Pantoea sp. and Mixta sp. Isolates produced significant amount of Indole-3-acetic acid (IAA) (Enterobacter hormaechei BHUJPCS-15; 58.91 μg/ml), solubilised phosphate (Bacillus subtilis BHUJPCS-24; 999.85 μg/ml) and potassium, ammonia (Bacillus subtilis BHUJPCS-12; 148.73 μg/ml), and also inhibited the growth of chickpea pathogen (Pseudomonas aeruginosa BHUJPCS-7 against Fusarium oxysporum f.sp. ciceris) under laboratory conditions. Several seed endophytes induced significant increase in plant growth and increased tolerance of chickpea plants to the pathogen (Fusarium oxysporum f.sp. ciceris) when tested in vitro. Re-introduction of these isolates, resulted in significant increase in plant length, biomass and chlorophyll contents and bio-controlling activity against Fusarium oxysporum f.sp. ciceris. These results provide a direct evidence for the presence of beneficial seed microbiome and suggest these isolates could be further developed into potential bio-inoculants for improving diseases management and sustainable increase in agriculture productivity.

Nicotiana tabacum seed endophytic communities share a common core structure and genotype-specific signatures in diverging cultivars

Seed endophytes of crop plants have recently received increased attention due to their implications in plant health and the potential to be included in agro-biotechnological applications. While previous studies indicated that plants from the Solanaceae family harbor a highly diverse seed microbiome, genotype-specific effects on the community composition and structure remained largely unexplored. The present study revealed Enterobacteriaceae-dominated seed-endophytic communities in four Nicotiana tabacum L. cultivars originating from Brazil, China, and the USA. When the dissimilarity of bacterial communities was assessed, none of the cultivars showed significant differences in microbial community composition. Various unusual endophyte signatures were represented by Spirochaetaceae family members and the genera Mycobacterium, Clostridium, and Staphylococcus. The bacterial fraction shared by all cultivars was dominated by members of the phyla Proteobacteria and Firmicutes. In total, 29 OTUs were present in all investigated cultivars and accounted for 65.5% of the combined core microbiome reads. Cultivars from the same breeding line were shown to share a higher number of common OTUs than more distant lines. Moreover, the Chinese cultivar Yunyan 87 contained the highest number (33 taxa) of unique signatures. Our results indicate that a distinct proportion of the seed microbiome of N. tabacum remained unaffected by breeding approaches of the last century, while a substantial proportion co-diverged with the plant genotype. Moreover, they provide the basis to identify plant-specific endophytes that could be addressed for upcoming biotechnological approaches in agriculture.

Priority effects of wheat seed endophytes on a rhizosphere symbiosis

Seed endophytes, as primary symbionts, may influence formation of secondary symbioses in maturing hosts. We hypothesize that such priority effects are a function of the presence and the identity of the primary symbiont, and are simulated via inoculation with primary symbionts. We sampled primary symbionts in winter wheat using culture-based techniques and next generation sequencing. Ninety-one percent of 6300 seeds had either one or no isolates. NGS data supported this with over 99% fewer reads detected in seeds having no isolates. Alternaria and Cladosporium spp., accounted for almost 80% of the primary symbionts isolated from seeds but only 36% of OTUs. Bacteria accounted for 13% of isolates but 27% of OTUs. We grew seedlings with or without primary symbiont isolates aseptically in vitro with roots in contact with a suspension of the rhizosphere symbiont Phialocephala fortinii. Seedlings having Cladosporium spp. or bacteria as primary symbionts increased the P. fortinii root colonization in 3 weeks by 30% and 50%, respectively, compared to seedlings lacking symbionts. Alternaria primary symbionts had no effect on root colonization by P. fortinii. However, Cladosporium and Alternaria inocula reduced P. fortinii colonization by 75% and 42%, respectively, at 3 weeks, so natural priority effects were not simulated by inoculation with fungi. Bacillus inoculum doubled P. fortinii colonization, but colonization was delayed. Primary symbionts of wheat seed can have strong, natural priority effects dependent on their presence and identity, but inoculation does not always simulate natural effects.

A Hypothetical Bottleneck in the Plant Microbiome.

The plant microbiome may be bottlenecked at the level of endophytes of individual seeds. Strong defense of developing seeds is predicted by optimal defense theory, and we have experimentally demonstrated exclusionary interactions among endophytic microbes infecting individual seeds of Centaurea stoebe. Having found a single, PDA-culturable microbe per seed or none in an exploratory study with Centaurea stoebe, we completed a more extensive survey of an additional 98 plant species representing 39 families. We again found that individual, surface-sterilized seeds of all species hosted only one PDA-culturable bacterial or fungal endophyte per seed, or none. PDA-unculturables were not determined but we expect them to also be bottlenecked in individual seeds, as they too should be governed by exclusionary interactions. If the bottleneck were confirmed with high-throughput sequencing of individual seeds then it would make sense to further investigate the Primary Symbiont Hypothesis (PSH). This includes the prediction that primary symbionts (i.e., the winners of the exclusionary battles among seed endophytes) have strong effects on seedlings depending on symbiont identity.

Molecular identification and characterization of endophytes from uncultivated barley

ABSTRACTEpichloë species (Clavicipitaceae, Ascomycota) are endophytic symbionts of many cool-season grasses. Many interactions between Epichloë and their host grasses contribute to plant growth promotion, protection from many pathogens and insect pests, and tolerance to drought stress. Resistance to insect herbivores by endophytes associated with Hordeum species has been previously shown to vary depending on the endophyte-grass-insect combination. We explored the genetic and chemotypic diversity of endophytes present in wild Hordeum species. We analyzed seeds of Hordeum bogdanii, H. brevisubulatum, and H. comosum obtained from the US Department of Agriculture’s (USDA) National Plant Germplasm System (NPGS), of which some have been reported as endophyte-infected. Using polymerase chain reaction (PCR) with primers specific to Epichloë species, we were able to identify endophytes in seeds from 17 of the 56 Plant Introduction (PI) lines, of which only 9 lines yielded viable seed. Phylogenetic analyses of housekeeping, alkaloid biosynthesis, and mating type genes suggest that the endophytes of the infected PI lines separate into five taxa: Epichloë bromicola, Epichloë tembladerae, and three unnamed interspecific hybrid species. One PI line contained an endophyte that is considered a new taxonomic group, Epichloë sp. HboTG-3 (H. bogdanii Taxonomic Group 3). Phylogenetic analyses of the interspecific hybrid endophytes from H. bogdanii and H. brevisubulatum indicate that these taxa all have an E. bromicola allele but the second allele varies. We verified in planta alkaloid production from the five genotypes yielding viable seed. Morphological characteristics of the isolates from the viable Hordeum species were analyzed for their features in culture and in planta. In the latter, we observed epiphyllous growth and in some cases sporulation on leaves of infected plants.