The presence of a significant endophytic fungus in mycobiome of rice seed compartments

doi:10.21203/rs.3.rs-4019379/v1

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The presence of a significant endophytic fungus in mycobiome of rice seed compartments

https://doi.org/10.21203/rs.3.rs-4019379/v1

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published 08 Oct, 2024

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Seed microbial communities have been known to have a crucial role in the life cycle of a plant. In this study, we examined the distribution of the fungal communities in three compartments (husk, brown rice, and milled rice) of the fourteen rice seed samples using Illumina MiSeq sequencing. A total of 894 fungal operational taxonomic units were found at 97% sequence identity, and ten fungal genera distributed throughout the three compartments of the rice seeds were identified as the core mycobiome of the rice seed. Based on the diversity analysis, the distribution of the fungal community in milled rice was found to be more diversified, evenly distributed, and differently clustered from the other two compartments. Among core mycobiome, Moesziomyces dominated almost 80% of the fungal communities in the outer compartments of rice seeds, whereas the abundances of other endophytic pathogenic fungi declined. Our results provide that antagonistic yeast Moesziomyces may be able to control the endogenous pathogenic fungal communities in rice seeds, hence maintaining the quality of rice seeds. In addition, the distribution of fungal communities differs depending on the rice seed’s compartment, indicating that the compartment can affect the distribution of the seed microbial community.

Biological sciences/Microbiology

Biological sciences/Microbiology/Communities

Biological sciences/Microbiology/Fungi

Rice seed mycobiome

seed compartment

husk

brown rice

milled rice

Moesziomyces

The plant microbiome—the plant-associated microbial community—performs a variety of important functions related to the development, health, and productivity of the host plant [1]. The communities of microbes in plants play a vital role in enabling host plants to adapt to abiotic stress [2, 3], enhance nutrient absorption [4, 5], and stimulate defense mechanisms against plant pathogens [6, 7]. Among the plant microbiome, the rhizosphere, owing to harboring a multitude of microbial species from the surrounding soil, is the prominent focus of research within the plant microbiome [8]. Researchers have recently become increasingly aware of the importance of microbes that live on the plant’s surface (epiphytes) and within it (endophytes) for protecting against pathogens and assisting the host’s development and production [9–11]. Consequently, a considerable amount of research has been conducted to characterize the plant microbiome of various plants [12, 13].

The seed is the starting point for symbiotic connections between plant and microbial communities [14]. The seed microbiome was likely to be transmitted to the next generations, connecting one generation to the next and influencing the phenotype and survival of future plants [15–17]. Endophytes present in internal tissues of the seed, such as the endosperm and embryo tissue, are transferred vertically to the next generation and form the microbial communities of the future plants from the early development stage [12, 18]. Seed epiphytes, on the other hand, are in contact with the external environment, so their composition is thought to vary depending on the various biotic and abiotic factors [19, 20]. However, the seed epiphytes can become endophytes by entering the seed through the small pores of the seed coat, and vice versa [18]. Also, the epiphytes can provide the first host defense against external pathogens and antagonize other species with in the microbial population [21, 22]. Therefore, understanding the epiphytic and endophytic seed microbiome can be expected to provide insight into the formation and interactions of the seed microbiome [23, 24].

Rice (Oryza sativa) is a primary food staple for a large population, mostly in the Asian region [25, 26], and rice microbiome research has been conducted alongside wheat and maize, which together represent the three major plant microbiome investigations [27]. The primary emphasis of rice microbiome research has been on the examination of microbial communities in the rhizosphere of rice [28, 29]. These studies revealed that these microbial communities have a beneficial role in supporting rice plants by facilitating nutrient delivery and absorption, enhancing plant resilience against stress, and promoting growth [30]. Next to the rhizosphere, considerable study has been conducted on the rice seed microbiome, the reproductive organs of rice plants [31–33]. The significance of relevant study is emphasized due to the role of rice seeds in shaping the microbiome throughout the entire life cycle of rice through vertical transmission to the next generation [8, 34]. Furthermore, while most plant microbiome research focuses on the bacterial population, additional study on the fungal community of rice is also emphasized [27, 31].

The focus of this research was to compare the fungal compositions of rice seed separated into three distinct compartments, the husk, brown rice, and milled rice. Fourteen unpolished rice seed samples from eight different regions of South Korea was collected and divided into three compartments through dehulling and milling processes. Following a 24-hour immersion in buffer of three compartments of the rice seeds to extract both endophytes and epiphytes, the microbial DNA were extracted from each, however only single replicates were obtained. Fungal profile was constructed by amplifying the internal transcribed spacer (ITS) 1 region using Illumina MiSeq sequencing techniques. Our results showed that the proportion and diversity of the fungal communities differs between milled rice, which is the inner most part of the rice seed, and the husk and brown rice, which contains the bran compartment of the rice seed. We also found that the endophytic yeast Moesziomyces accounts for more than 75% of the fungal profile in both husk and brown rice. In milled rice, the proportion of Moesziomyces decreased while the diversity and evenness of other fungi rose. In this study, we compared the fungal communities in three compartments of fourteen rice seed samples to determine the distribution of epiphytic and endophytic fungi in rice seeds. Furthermore, our findings of a significant abundance of the endophytic yeast Moesziomyces, which has antagonistic activity against plant pathogens, in the exterior compartments of rice seed can be used in the development of microbial-based protection against plant pathogens.

The fungal community compositions of three compartments of the rice seed: husk, brown rice, and milled rice

To compare the fungal communities in three compartments of the rice seed, we constructed fungal profiles of three distinct compartments of fourteen rice seed samples, namely the husk, brown rice, and milled rice (Fig. 1) through Illumina MiSeq. From 77,258 to 148,666 high-quality ITS1 fungal reads were generated and a total of 894 fungal operational taxonomic units (OTUs) were determined at 97% sequence identity (Table 1). A total of 894 fungal OTUs were classified into 3 phyla, 21 classes, 68 orders, 141 families, 211 genera, and 265 species, excluding the unknown and unclassified ones (Supplementary Table S1). The average number of fungal OTUs found in husk, brown rice, and milled rice was 97, 141, and 121, respectively. The rarefaction curves for richness in all three compartments of rice seeds plateau between 3,500 and 5,000 reads (Fig. 2), proving that the sequencing depths of all samples were sufficient. There was noticeable difference in the reaching the asymptote point between milled rice and the other two groups. The fungal rarefaction curve of milled rice reached the asymptote faster than that of husk and brown rice, indicating that the fungal richness of milled rice is higher than other two groups.

Table 1

The list of rice seed samples analyzed in this study.
No.	Rice seed^a	No. of total reads^b			No. of total OTUs^b			Sample origin
No.	Rice seed^a	Husk	Brown rice	Milled rice	Husk	Brown rice	Milled rice	Sample origin
1	CW1	110,326	117,866	92,466	74	129	103	Cheorwon, Gangwon
2	CC1	99,645	140,267	97,640	98	43	61	Chuncheon, Gangwon
3	CC2	109,182	121,251	77,258	103	188	111	Chuncheon, Gangwon
4	SW1	90,866	101,445	99,277	94	60	96	Suwon, Gyeonggi
5	SW2	104,148	138,844	121,164	78	211	125	Suwon, Gyeonggi
6	HS1	78,997	88,050	114,553	72	65	90	Hwaseong, Gyeonggi
7	HS2	105,541	113,858	91,330	113	184	154	Hwaseong, Gyeonggi
8	SJ1	96,290	148,666	95,412	143	51	126	Sangju, Gyeongbuk
9	SJ2	96,362	100,349	101,097	103	187	147	Sangju, Gyeongbuk
10	MY1	91,499	110,787	96,400	117	190	146	Miryang, Gyeongnam
11	JJ1	92,178	137,948	78,663	71	223	69	Jeonju, Jeonbuk
12	JJ2	87,946	112,977	114,488	98	198	167	Jeonju, Jeonbuk
13	CJ1	94,569	87,370	79,687	67	70	127	Cheongju, Chungbuk
14	CJ2	114,920	116,683	97,842	123	170	173	Cheongju, Chungbuk
^aFourteen rice seed samples were collected from eight locations in Korea. The collected rice seeds were processed into three compartments (husk, brown rice, and milled rice) through the dehulling and milling process.
^bMycobiome analysis of three compartments of the rice seed were performed using Illumina MiSeq platform. The total number of reads was 77,258 to 148,666, and the total number of OTUs was 43 to 223.

Diversity of the fungal communities of three compartments of the rice seed

Alpha diversity of the fungal communities in three compartments of the rice seed was measured by observed fungal OTU richness, Chao1 index, Shannon index, and Simpson index (Fig. 3). The number of observed fungal OTUs in husk, brown rice, and milled rice ranged from 26 to 38 and increased in the inner compartments of the rice seed (Fig. 3a). Among the three compartments of the rice seed, there was a significant difference in the number of observed fungal OTUs in husk and milled rice (P = 0.009). The Chao1 index was also increased in the inner compartments of the rice seed, and was considerably higher in milled rice than husk (P = 0.005). Both observed fungal OTU richness and Chao1 index significantly increased from husk to milled rice, the latter harbored a considerable fungal community. Based on the Shannon index, there was no significant difference of the diversity of fungal communities between husk and brown rice (P = 0.270), but milled rice exhibited more diversity than husk (P = 9e-10) and brown rice (P = 6e-10) (Fig. 3b). Moreover, milled rice had a more even distribution of fungal composition than husk (P = 1e-08) and brown rice (P = 3e-12). To summarize, these findings indicate that the diversity and evenness of interior fungal communities of rice seed is greater than that of the exterior.

To better understand the distribution of fungal communities in three compartments of rice seeds, PCoA with the Bray-Curtis dissimilarity matrix was used to find out the internal relationships of fungal communities of the husk, brown rice, and milled rice (Fig. 4). PCoA matrix showed that fungal communities clustered generally based on three distinct compartments of rice seeds. The fungal PCoA for two compartments, husk and brown rice, overlapped, indicating that fungal communities associated with husk and brown rice were similar. On the other hand, the fungal PCoA of milled rice clustered very distinctly from the husk and brown rice (Fig. 4). These results indicate the statistical differences of the diversity of fungal communities in milled rice and the outer compartments that surround it.

Taxon composition of the fungal communities of three compartments of the rice seed

A total of 894 fungal OTUs were primarily detected and 3 phylum, 211 genera and 265 species were identified (Supplementary Table S1). The two main phyla detected in rice seed were Ascomycota and Basidiomycota, with other minor taxa including Mucoromycota, and unknown or unclassified fungi (Supplementary Table S2). The Basidiomycota phylum has the highest percentages in husk and brown rice with 79.8 ± 12.8% and 78.3 ± 9.2%, respectively, followed by Ascomycota with 19.3 ± 12.8% and 21.1 ± 8.9% (Fig. 5a). In milled rice, on the other hand, the Ascomycota phylum has the highest percentages with 62.9 ± 14.6%, followed by Basidiomycota with 36.7 ± 14.4%.

Ten of the 211 genera had an abundance of more than 1% in at least one sample (Fig. 5b, Table 1). Among ten genera, Moesziomyces was the most abundant genus in all rice seed compartments, with an overall abundance ranging from 17.6% (milled rice of SW1 rice seed sample) to 90.3% (husk of CJ1 rice seed sample). In husk and brown rice, Moesziomyces was the predominant genus, accounting for 69.8 ± 12.3% and 76.6 ± 9.6%, respectively. In contrast, the abundance of Moesziomyces in milled rice was significantly lower than in husk (P = 4e-07) and brown rice (P = 7e-09), accounting for 35.8 ± 14.2%. Two Moesziomyces species (M. antarcticus and M. aphidis) were identified, with M. antarcticus being the most abundant species of all compartments with an abundance of 14.8–90.0% (Supplementary Table S3). In addition to Moesziomyces, four genera showed significant differences of abundance between milled rice and the other two compartments of the rice seed (Table 2). The mean relative abundance of Alternaria, Epicoccum, Nigrospora, and Phaeosphaeria in milled rice is 15.2%, 11.3%, 6.9%, and 3.5 respectively, which is approximately three to five times higher than that of husk and brown rice (P < 0.01). The abundance of Neosetophoma in all three compartments was less than 5%, but there was significant difference of abundance between husk and milled rice (P = 0.001). Cladosporium and Fusarium were abundant in all three compartments of the rice seed (> 3.5%), but there was no statistically significant difference (Table 2). Cladosporium showed the highest abundance in husk accounting for about 7.7%, with the biggest difference with milled rice (P = 0.030). Fusarium showed the highest abundance in milled rice accounting for about 8.9%, with the biggest difference with husk (P = 0.045).

Table 2

Relative abundance and ANOVA statistics of the top 10 fungal genera in husk, brown rice, and milled rice.
Phylum	Genus^a	Relative abundance (%) ^b
Phylum	Genus^a	Husk	Brown rice	Milled rice
Basidiomycota	Moesziomyces	69.8 ± 12.3x	76.6 ± 9.6x	35.8 ± 14.2y
Ascomycota	Alternaria	0.7 ± 1.2x	3.1 ± 3.7x	15.2 ± 11.3y
Ascomycota	Epicoccum	2.3 ± 1.9x	2.7 ± 2.6x	11.3 ± 7.5y
Ascomycota	Cladosporium	7.7 ± 6.0x	4.1 ± 3.3x	3.7 ± 1.6x
Ascomycota	Fusarium	3.5 ± 3.9x	4.2 ± 8.4x	8.9 ± 8.5x
Ascomycota	Nigrospora	0.3 ± 0.4x	0.9 ± 0.8x	6.9 ± 4.7y
Ascomycota	Phaeosphaeria	0.2 ± 0.2x	1.1 ± 1.3x	3.5 ± 2.7y
Ascomycota	Setophoma	0.1 ± 0.1x	1.1 ± 1.9x	2.9 ± 4.4x
Ascomycota	Neosetophoma	0.1 ± 0.1x	1.2 ± 1.7xy	2.5 ± 2.2y
Ascomycota	Sarocladium	1.6 ± 2.5x	0.1 ± 0.2x	0.3 ± 0.3x
^aFungal genera showing significant differences between husk, brown rice, and milled rice are indicated in bold (P < 0.01).
^bValues are presented as the mean ± standard deviation. The average relative abundance (%) and ANOVA test statistics of fungal genera with the top 10 abundance in husk, brown rice, and milled rice are shown. Different letters in the same line indicate significant differences (P < 0.01) as determined by analysis of variance.

Nowadays, it is understood that a wide range of internal and external factors influence how plants’ microbiome is shaped [35–37]. Environmental elements such as soil, weather and moisture influence the formation of plant microbiomes, but it’s unclear how much influence each has on the plant microbial population [38, 39]. Also, most research on plant microbiome focuses on soil and roots [29, 40], there is little microbiome study on seeds [14, 41]. In this study, we examined the fungal distribution of rice seeds and how it was distributed across three distinct compartments of rice see; husk, brown rice, and milled rice (Fig. 1). To elucidate whether rice seeds have a core mycobiome across compartments, we collected fourteen rice seeds from seven different provinces of Korea (Table 1). Each rice seed sample was dehulled and milled, resulting in three compartments: husk, which was the outermost layer, brown rice, which contained the bran layer, and milled rice which removed the bran and aleurone layer (Fig. 1a). To identify all endophytes and epiphytes of three compartments of the rice seeds, the steeped solution was used to extract total microbial DNA after immersing each sample in buffer for a day. Mycobiome analysis was then performed on three compartments of fourteen rice seed samples (Fig. 1b). The rarefaction curve of generated reads reached the asymptote, indicating that enough sequencing had been done to support additional investigation (Fig. 2).

Previous studies have shown that seeds have particularly low microbial biomass and diversity compared to other plant compartments and soil [15, 42]. However, our findings indicated that the diversity and evenness of the fungal distribution vary depending on the compartment of the rice seeds. The results of the diversity analysis showed high abundance, diversity, and evenness of the fungal communities identified in milled rice from which the outermost layer of the rice seed (husk) and the next layer (bran) have been removed (Fig. 3). In comparison to the other two compartments, the milled rice exhibited more fungal richness as determined by the total number of fungal OTUs and the Chao1 diversity index (Fig. 3a). In addition, the milled rice exhibited a notably higher Shannon index and Simpson index than the other two compartments, indicating that more diverse fungal communities were distributed evenly in milled rice (Fig. 3b). The high alpha diversity index of milled rice indicated that the diverse fungal communities are evenly distributed in the rice seed’s inner compartment, while the outer compartment is dominated by a small number of fungal communities. Moreover, the beta diversity indices revealed that the milled rice had a distinct clustering pattern, although the husk and brown rice samples exhibited some degree of overlap (Fig. 4). Our findings suggest that the seed compartments had a significant impact on shaping of rice seed microbiome [31, 41, 43].

To investigate the mycobiome distribution of the rice seeds, we compared the distribution of fungal communities in three compartments of rice seeds at the phylum and genera levels (Fig. 5, Table 2). The major phylum seen in the husk and brown rice was Basidiomycota, which accounted for almost 80% of the total (Fig. 5a), consistent with its abundance in the rice phyllosphere (77 ~ 100%) reported in previous studies [31, 44]. The majority of the Basidiomycetes found in the husk and brown rice were identified as Moesziomyces, which is widely distributed in the cereal crops and Poaceae family, particularly rice in Eastern Asia [45–47]. Because Moesziomyces constitutes the majority of the fungal community in the outer compartments of the rice seeds, the fungal distribution in the husk and brown rice is less diverse and uneven. In contrast, the abundance of Moesziomyces in the milled rice reduced to 35% while the Ascomycota fungi increased to about 63% (Fig. 5b, Supplementary Table S2). Ascomycetes such as Alternaria, Epicoccum, Nigrospora, and Phaeosphaeria have increased three to five-fold and Fusarium and Cladosporium accounted for high proportions of 8.9% and 3.7% in milled rice, respectively. As these genera become more abundant in milled rice, the diversity and evenness of the fungal communities in milled rice is thought to be rather higher than in the outer compartments.

The fungal communities of three compartments of rice seed showed significant taxonomic overlap, and the core mycobiome of ten genera distributed with high abundances across the three compartments was identified (Table 2). Moesziomyces was the most prevalent fungus found in all three compartments of the rice seeds which can be frequently obtained from the phyllosphere of numerous plants [45, 48–50]. Among the other core mycobiomes of rice seeds, Alternaria, Epicoccum, and Fusarium are known to be the most common plant-pathogenic fungi [51–53]. The innermost compartment of the rice seed, milled rice, had a relatively high abundances of those plant-pathogenic fungi, suggesting the possibility of seed-borne disease. Seeds may act as carriers of plant-pathogenic fungi that affect growth and productivity of growing plants when environmental conditions and other cultural characteristics are suitable [54, 55]. Thus, pathogenic fungi found in the seeds are one of the potential threats to seed quality and crop productivity [56–57]. However, our results showed that the distribution of plant pathogenic fungi varied depending on the compartment of the rice seeds and tended to be adversely correlated with the distribution of Moesziomyces (Table 2). Moesziomyces, formerly known as Pseudozyma, has been known as a biocontrol yeast against several plant-pathogenic fungi [58–59]. A previous study found that Pseudozyma strains inhibit the growth of fungal pathogens via antibiosis and ectoparasitism [60] and could protect crops from both bacterial and viral infections [50]. Also, Moesziomyces is known as an antagonistic yeast that suppresses the growth of Fusarium strains, which causes FHB in cereal crops [61, 62]. In this study, we found that Moesziomyces had a high distribution on the exterior compartments of the rice seeds, dominating the distribution of other pathogenic fungi. Our findings, as well as previous researches, indicate that Moesziomyces is an efficient endogenous yeast found in rice seed that can reduce seed-borne plant diseases.

Seeds harbor a wide range of microorganisms that can be transmitted to growing plants. Thus, seed endophytic microbes play a significant influence in seed quality and the occurrence of seed-borne plant diseases. The core mycobiome of rice seeds discovered in this study showed relatively high abundances of pathogenic fungi that may act as potential pathogens. However, Moesziomyces, an antagonistic epiphytic yeast, dominates the fungal communities of outer compartment of rice seeds, so it is thought to be able to maintain the quality of rice grains by inhibiting the growth of pathogenic fungi. Moreover, our findings give insight that the composition and distribution of mycobiome vary throughout the compartments of rice seeds, irrespective of the region where the rice seeds were collected. The primary determinants of the microbial community of plants are usually known as the environmental conditions and genetic variations within the host plants [39]. However, our study showed that the compartments of rice seed can also be a key driver in shaping the microbial communities.

Collection of three compartments of the rice seed samples

Fourteen rice seed samples harvested in 2018 from eight provinces of South Korea were collected by the National Institute of Crop Science (South Korea). Each rice seed was separated into husk and brown rice through the dehulling process, and into white rice through a milling process that removes bran and aleurone layers of brown rice (Fig. 1a) [63]. A total of forty-two samples including husk, brown rice (hulled rice or unmilled rice), and milled rice (white rice) of fourteen rice seeds (Fig. 1b) were obtained from the National Institute of Crop Science (South Korea). Each sample was stored at -20°C until DNA extraction.

The use of rice seed samples was in accordance with all relevant guidelines and regulations. We received permission from the National Institute of Crop Science before collecting rice seed.

DNA extraction and sequencing

Fifty grams of three compartments of the rice seed samples were soaked in 200 ml of phosphate-buffered saline (PBS), incubated at 4 ˚C for 24 hours, and incubated at 25 ˚C with 180 rpm shaking for 1 hour. The culture was harvested and stored at -20°C until DNA extraction. The fungal DNA was extracted from the pellet using the PowerSoil gDNA extraction kit, following the manufacturer’s instructions. Then, the concentration and quality of gDNA were assessed using Nanodrop (Thermofisher, USA). In order to identify the fungal community, the ITS1 region from the rDNA was amplified using the universal primers ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS2 (5’-GCTGCGTTCTTCATCGATGC-3’) [64, 65]. The PCR was performed under the following conditions: initial denaturation at 95 ˚C for 3 min, then 25 cycles of 95 ˚C for 30 sec, annealing at 55 ˚C for 30 sec, and extension at 72 ˚C for 30 sec, with a final elongation at 72 ˚C for 5 min. The amplicons were sequenced by MiSeq (Illumina, San Diego, CA, USA) in accordance with the service manuals provided by Macrogen (Seoul, Korea).

Data processing and analysis

The raw reads were first demultiplexed, and FastQC Version 0.11.9 was used to assess the quality of the reads. The LotuS pipeline [66] was used for filtering and clustering the fungal ITS sequences based on the UNITE database [67]. The clustered sequences were denoised at the 97% identity level to generate operational taxonomic units (OTUs). The NCBI nucleotide (nr/nt) database was used to identify the OTUs, and the taxonomy was determined using the ETE 3 toolkit [68]. The level of taxonomic assignment was determined by the Lotus pipeline’s default parameters if the best hit similarity was less than 97%. These settings were 97% for species, 95% for genus, 93% for family, 91% for order, 78% for class, and 97% for phylum. To identify the distribution of fungal taxa across the three compartments of rice seeds, that accounted for at least % of the total reads in at least one sample were analyzed. And the data was processed using phyloseq [69] and microbiomeutilities in the R package (https://microsud.github.io/microbiomeutilities/). The rarefaction curves for samples were calculated to a depth of 5,000 reads using the Rarefy in R package (https://arts.units.it/handle/11368/2966451). The richness and diversity were calculated based on the species count table, and the differences in the alpha-diversity indicators (Observed, Chao1, Shannon, and Simpson) between samples were estimated using Kruskal-Wallis nonparametric analysis of variance followed by Dunn’s posthoc test. The principal coordinate analysis (PCoA) was used for the analysis of beta-diversity through the coordination of Bray-Curtis distances.

Data availability

The mycobiome data of three compartments of rice seeds have been deposited to the NCBI Sequence Read Archive database under the accession numbers SRR14671889 - SRR14671930.

Acknowledgments

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Crop Viruses and Pests Response Industry Technology Development Program (Grant Number: 320036-5), which is funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA), and the “Cooperative Research Program for Agriculture Science and Technology Development (RS-2023-00230782)", which is funded by the Rural Development Administration, Republic of Korea.

Author contributions

J.A.S. conceived the project. E.J., and N.A. carried out experiments and validation. N.A. and J.Y.L performed mycobiome analysis. E.J, and J.A.S. wrote the manuscript and revised the manuscript. All authors read and approved the manuscript.

Competing interests

The authors declare no competing interests.

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No competing interests reported.

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The presence of a significant endophytic fungus in mycobiome of rice seed compartments

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Abstract

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Introduction

Results

Diversity of the fungal communities of three compartments of the rice seed

Taxon composition of the fungal communities of three compartments of the rice seed

Discussion

Materials and methods

Collection of three compartments of the rice seed samples

DNA extraction and sequencing

Data processing and analysis

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References

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Supplementary Files

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