Introduction

Endophyte communities are found in almost all plants [1, 2]. These communities usually consist mainly of bacteria or fungi [3] that reside inside plant organs and do not damage the host tissue [3–7]. Some fungal endophytes can spend part of their life cycle as latent pathogens in plant tissues [8–10]. Fungal endophytes are vital in enhancing plant resilience against various stresses, such as abiotic factors like drought and biotic factors like pathogens and pests [11–13].

The presence of endophytes in plant tissues is typically determined using microscopic and media-cultivation methods [14–16]. However, DNA-based methods have become increasingly implemented [17]. PCR has been employed to detect endophytes in various grass tissues, including Epichloë, Neotyphodium, and Acremonium [18–21]. Microdochium bolleyi and Microdochium phragmitis were detected by nested PCR [22]. In addition to PCR, alternative methods such as PCR-DGEE (polymerase chain reaction and denaturing gradient gel electrophoresis) [23], Droplet Digital PCR Technology [24], microarray [25], sequencing [26], and immunological tests [27] can be employed to detect endophytes and other fungi residing in plant tissues. Furthermore, modern "omics" techniques such as comparative genomics, metagenomics, and metatranscriptomics have the potential to detect endophytes and unravel the relationships between plants and endophytes [28].

Microdochium bolleyi (Mb) is an endophytic fungus that primarily colonizes roots of cereals and grasses, including wheat [29], barley [30, 31], Brachypodium distachyon (Bd) [32], and others [33–35]. Mb has been observed to suppress various plant pathogens affecting cereals [29, 31, 36–39]. The formation of typical and under light microscopy visible chlamydospore clusters within colonized plant cells makes this endophytic fungus an excellent model organism to study plant endophyte interactions [32].

The first objective of this study was to develop species-specific primers for qPCR detection and quantification of endophyte M. bolleyi in plant tissues and thorough testing of their specificity and capability of detecting the endophyte in plants. The second aim of the study was to assess the colonization dynamic over time, the location of the endophyte in plants, and the seed transmission potential of the endophytic fungus M. bolleyi using wheat and the model plant species B. distachyon.

Materials and methods

Biological materials

All plants were cultivated in a cooled greenhouse (20/18°C, day/night) within pots 8 cm in diameter filled with a 50:50 planting substrate–sand (vol:vol) mixture, the substrate FLORCOM SV (BB Com, Letohrad, Czech Republic). For the experiment were used not treated seeds of the wheat cultivar Bohemia and of B. distachyon line Bd21 obtained from the Joint Genome Institute (https://jgi.doe.gov). Ten seeds were placed into each pot. After germination, the number of plants in every pot was reduced to six.

Six Mb isolates were used (UPOC-FUN-253–258) from the Collection of Phytopathogenic Microorganisms UPOC (Czech Republic). Inoculum of Mb isolates was obtained by cultivation on double sterilized millet seed [32], which was previously tested for the absence of Mb by qPCR. The inoculation of the plants was also carried out according to the method of Matušinsky et al. (2022) by adding inoculum to the seeds during the sowing [32]. Only sterile millet without endophyte was added to the control treatments.

Isolates of other fungi used for testing primers were obtained from four different collections of microorganisms (Table 1).

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Table 1. Fungal species used in the analysis with the MbqITS primers designed in this study.

A positive reaction result is indicated by a Cq lower than 30. The Cq levels in the table are the average of three replicates.

https://doi.org/10.1371/journal.pone.0297633.t001

Results

Demonstrated root colonization by Mb of wheat and Bd

Using microscopic and molecular methods, the presence of Mb was detected in both wheat and Bd samples. Using light microscopy, chlamydospores were observed in the seed coats, roots, crowns, and stem bases of wheat and Bd (S2, S3 Tables and Fig 1). No presence of chlamydospores was observed in plants without inoculation.

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Fig 1.

Chlamydospores of Microdochium bolleyi in wheat leaf sheaths (A, B) and comparison of seed coats of non-inoculated (C) and inoculated (D) Brachypodium distachyon. Scale bar = 100 μm.

https://doi.org/10.1371/journal.pone.0297633.g001

A set of MbqITSF/R best met the specificity requirements of the five designed primer pairs. The primers showed positive results in all six tested isolates of Mb (S4 Table). In the specificity test, the best-selected primers amplified DNA only from Mb, not any other fungal pathogens screened (Table 1). The primer set was therefore used to examine Mb-inoculated and non-inoculated plant material (wheat and Bd). Inoculated plants with chlamydospores present in plant tissues of both wheat and Bd showed positive results after qPCR with primers MbqITSF/R and, on the contrary, non-inoculated plants without chlamydospores in plant tissues showed negative results with Cq values higher than 30. The analysis confirmed that the primers were specific and robust in detecting and quantifying Mb within plant tissues. No false positive results were seen after melting analysis. Other tested primer pairs, MbqLSU1F/R, MbqLSU2F/R, MbqPOL1F/R, and MbqPOL2F/R, failed in the specificity test and were excluded from other parts of this study. The efficiency of the reaction was determined using a dilution series of DNA from the Mb isolate (UPOC-FUN-253). Based on the standard curve obtained, the efficiency (E) of the reaction was found to be 98.5% (S5 Table). The method showed a reliable positive response at 0.001 ng of Mb DNA (S5 Table), and the melting curve gives a single peak, indicating no artifacts.

Dynamics of Mb colonization in wheat and Bd over time

The seed coats, i.e., the already dead tissue, are massively colonized (30 days after inoculation) (Fig 1C and 1D). Only then are the living parts of the plant inhabited, such as the crown and roots, especially the part of the roots near the crown (30 dai). Over time (90 dai), the endophyte spreads to the distal parts of the roots and in wheat to the higher parts of the stem, 2–4 cm, but in Bd, only up to 1 cm and not higher. The DNA of the endophyte increases over time in the aerial parts, in wheat earlier than in Bd (Fig 2). In the underground parts, especially in the lower distal part of the roots, however, the level of endophyte DNA is growing very slowly during sampling periods (30–150 dai) with no significant changes (Fig 2). In the wheat above-ground parts, the presence of chlamydospores is confirmed microscopically in the outer sheath of the oldest leaf sheaths enclosing the stem (60–90 dai). Gradually, chlamydospores can be found in the sheaths of the second-oldest and third-oldest leaves (120–150 dai). Usually, the sheaths of leaves already in the senescence stage are colonized in this way, so a tendency for Mb to inhabit sheaths already in the senescence stage can be observed. On microscopy, it was found that most of the chlamydospores in the aerial part of the plants are in the cells adjacent to the vascular bundles (Fig 1A and 1B). Still, the fungus does not penetrate the vascular bundle itself, and the tissues are free of visual symptoms, i.e., without browning or other signs of damage. At the last sampling date of 150 days after sowing, the plants were already completely dry and dead, and the increase in Mb DNA in crown and roots 1cm above crown in wheat and stem base 2–4 cm above crow in Bd is, therefore, more likely due to the saprotrophic effect of the fungus. No Mb DNA was detected in leaf blades, peduncles, ears, and harvested seeds, nor was the presence of chlamydospores seen in these parts of the plants (S2 and S3 Tables).

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Fig 2.

Dynamics of Microdochium bolleyi colonization in (A) wheat and (B) Brachypodium distachyon over time analyzed by qPCR. Statistically significant differences are indicated by asterixis (post hoc Tukey’s test, P < 0.05).

https://doi.org/10.1371/journal.pone.0297633.g002

Discussion

The fungal endophyte of grasses and cereals Mb is a suitable model organism for studying endophyte interactions with plants. Mb can colonize several cereal species, including wheat [29], barley [30], and the model organism Bd [32]. Molecular methods for detecting Mb have previously been described by nested PCR [22] and standard PCR [32]. In the current study, newly developed primers for qPCR were designed and tested to quantify the amount of Mb DNA in host tissues. The colonization dynamics over time, the distribution of endophytes in different parts of the plant, and the potential of seed transfer, i.e., vertical transfer, were tested in wheat and Bd. Although microscopic methods have been used to determine the presence of endophytes, in many cases, they are combined with molecular or other methods [31, 46, 47]. However, light microscopy could be time-consuming and inaccurate compared to molecular techniques. Identifying individual organisms using microscopy requires considerable experience, as species can only be distinguished based on morphological characters in this way. In addition, individual roots are usually not evenly colonized along their entire length, so only a particular part of the roots needs to be selected for microscopy. If the roots are too thick, focusing the slide in all aspects is challenging. Microscopic detection should be used as a companion tool with a more reliable and accurate molecular method but not as a stand-alone method for diagnosing endophytes.

Mb is a partially saprotrophic organism; in some cases, slightly pathogenic effects on wheat have been observed [30, 48]. The presence of Mb has been previously described in roots [30], crowns [49], and stems of cereals [50]. We used newly developed primers to screen the dynamics of Mb colonization of plants. First, massive colonization of the seed coats occurs, and only then are colonized roots near the crown and parts of the stem base within 1 cm of the crown. Over time Mb spreads to the distal parts of the roots and, in wheat, to the higher parts of the stem. In the lower parts of the roots, the level of endophyte DNA is more or less the same at all sampling periods without significant changes. Mb content in roots part 1 cm bellow crown increases in the last sampling term, e.g., 150 dai in wheat and 120 and 150 dai in Bd. In above-ground parts, the presence of chlamydospores is confirmed microscopically in the outer sheath of the oldest leaf enveloping the stem. No Mb DNA was detected in leaf blades, peduncles, ears, and seeds, nor was the presence of chlamydospores in these plant parts. Seeds from plants heavily colonized by Mb endophyte were harvested and sown, and plants from these seeds were examined for the presence of Mb by both microscopic and qPCR methods. No Mb endophyte was detected in any parts of the plants sown from these seeds, and therefore, it can be concluded that Mb is not transmitted vertically, i.e., by seeds.

Our study is not the first to address the quantification of endophytes in plant tissues using qPCR. For example, Chow et al. (2018) [51] used qPCR to investigate the effect of endophytes (Diaporthe phaseolorum, Trichoderma asperellum, and Penicillium citrinum) on the pathogen Ganoderma boninense that infects oil palm (Elaeis guineensis). The analysis showed that when plants were inoculated simultaneously with endophytes, and the pathogen G. boninense, colonization by the pathogen was suppressed by endophytic fungi, as reflected by the higher DNA content of endophytes compared to that of the pathogen. Latz et al. (2021) [52] investigated interactions between plant genotypes and the biotic and abiotic environment affecting the plant microbiome using the ITS1 metabarcoding method. Root microbial communities were less affected by abiotic factors than the phyllosphere microbiome. The diversity was highest in roots and lower in seeds, while leaves harbored the least diverse microbiome. The complete opposite was found in the work of Tao et al. (2008), who studied the distribution of fungal communities within leaf and root tissues of Bletilla ochracea (Orchidaceae) using random cloning, combined with DGGE (denaturing gradient gel electrophoresis) and phylogenetic analysis [53]. The main objective of their study was to compare the diversity of endophytes among roots and leaves of the same plants and to determine whether there is a consistency of communities within different organs of the same host. Fungal communities within leaf and root tissues differed significantly, and diversity within leaves was higher than within roots [53]. Bayman et al. (1997) used traditional isolation techniques and found that Xylaria spp. (Xylariales) and Rhizoctonia-like taxa (Basidiomycota) made up the majority of endophytes in epiphytic orchids, and fungal communities within leaves and roots were surprisingly similar [54].

These studies highlight the importance of conducting thorough investigations on endophytes and developing precise and reliable methods for detecting these organisms within the tissues of host plants. The use of molecular techniques in the determination of endophytes has wide applications. Cook et al. (2009) [55] used qPCR to determine the amount of an endophyte involved in producing a toxic alkaloid (swainsonine) in plants of the genus Oxytropis and Astragalus. The endophyte Undifilum oxytropis, involved in synthesizing the toxic alkaloid swainsonine, was quantified in plants of Oxytropis sericea and Astragalus mollissimus species using quantitative PCR. The amount of endophytes varies from one plant part to another and, in some cases, does not correspond to the concentration of swainsonine in the respective parts. The above works deal with microbial diversity inhabiting different parts of plants. Our work focused on one endophyte species and used qPCR to investigate the colonization dynamics and its distribution in the plant. The current study confirmed the endophyte presence in the roots and stem bases of wheat and Bd. Mb is found in roots, generally typical of the fourth class of endophytes to which this endophyte belongs [4]. Developing these newly designed primers has significantly broadened our understanding of the dynamics and distribution of endophytes within plants. Moreover, their application extends beyond our study, as they hold great potential for confirming successful colonization in commercial inoculations and facilitating further research in this field.

Fungal endophytes could be transmitted from plant to plant horizontally by soil or vertically by seed. A previous study by Sharon et al. (2023) [2] measured the correlation between fungal endophyte communities in seeds and stems of three cereal species under natural conditions and a controlled environment. Although most dominant fungal endophytic taxa could reach the seed internally, external seed infection (horizontal transfer) was found to be a primary source of specific taxa, including the most abundant species, Alternaria infectoria. Vertical transmission is not probably so frequent and is restricted to particular fungal taxa such as Epichloë [56]. Nevertheless, several studies indicate relatively more widespread vertical transfer than previously assumed [57, 58]. Mb belongs to the category of type 4 endophytes, and like other species in this group, it is recognized for its ability to be horizontally transmitted [4]. This has also been confirmed in our study. However, dead plants and debris remain on the ground and can be colonized by Mb. Murray and Gadd (1981) found that colonized seed remnants and roots remain in the ground after the crop is harvested and would provide a source of inoculum for the infection of barley seedlings in the following spring [30]. Regarding possible biological control, a potential approach could involve inoculation and colonization of seeds by Mb before sowing. By colonizing the seed coats, these cortical seed parts could serve as a source of inoculum and provide a favorable environment for seedlings’ inoculation. However, this hypothetical inoculation method has not yet been developed, and it would be helpful to test it in practice.

There are several reasons why the endophytic fungus Mb and plant Bd create an optimal model for studying plant-fungal interactions. The current study confirmed that the correspondence of Mb and Bd is excellent, and the fungus grows readily in Bd tissues. The inoculation method is simple, and the structures of Mb are readily visible in host tissues using light microscopy. In addition, we have developed and optimized species-specific primers for standard PCR [32] and here also for quantification of Mb by qPCR. The plant colonization dynamics by Mb are thoroughly studied in the current study. Previous studies have shown the beneficial effect of Mb on improving host plant immunity. Optimized methods and available primers from the last research [59, 60] allow the analysis of gene expression of specific marker genes in Bd. The advantage of Mb compared to strictly biotrophic symbiotic organisms is the possibility of cultivation on axenic media, and its massive sporulation ability makes its use suitable for large-scale production. Our results show that Bd and endophytic fungus Mb can be a model system for the plant-fungi interaction surveys. In general, a more profound knowledge of colonization dynamics and endophyte transmission could be used to improve agricultural management regarding plant growth promotion and biocontrol.

Conclusion

Mb is a suitable model for studying the interactions of endophytic fungi and plants. Primers for qPCR have been developed for the diagnosis and relative quantification of endophyte Mb in plant tissues and tested in wheat and Bd. The method is suitable for confirming the presence of endophytes in plants, studying colonization dynamics and distribution in plants, and other applications.

Supporting information

Acknowledgments

We thank the Collection of Phytopathogenic Microorganisms UPOC for providing fungal isolates.