Plant Biosystems - An International Journal Dealing with

all Aspects of Plant Biology

Official Journal of the Societa Botanica Italiana

ISSN: 1126-3504 (Print) 1724-5575 (Online) Journal homepage: https://www.tandfonline.com/loi/tplb20

Fungal diversity on the surface of saffron corms

with different growth characteristics

Shuwen Han, Qing Zhou, Jin Liu & Miao Da

To cite this article: Shuwen Han, Qing Zhou, Jin Liu & Miao Da (2020): Fungal diversity on the
surface of saffron corms with different growth characteristics, Plant Biosystems - An International
Journal Dealing with all Aspects of Plant Biology, DOI: 10.1080/11263504.2020.1739163

To link to this article: https://doi.org/10.1080/11263504.2020.1739163

Accepted author version posted online: 06

Mar 2020.

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Fungal diversity on the surface of saffron corms with different
growth characteristics

Shuwen Han1, Qing Zhou 2, Jin Liu3, Miao Da4,*

1
Department of Oncology, Huzhou Cent Hosp, Affiliated Cent Hops HuZhou University, 198 Hongqi

Rd, Huzhou, Zhejiang Province, China.

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2
Department of Nursing, Huzhou Central Hospital, No.198 Hongqi Rd, Huzhou, Zhejiang Province

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313000, China.

3
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Department of Pathology, Huzhou Central Hospital, No.198 Hongqi Rd, Huzhou, Zhejiang Province
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313000, China;
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4
Department of Nursing, Huzhou Third Municipal Hospital, No. 2088 East Tiaoxi Rd, Huzhou, Zhejiang Province
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313000, China;
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*
Corresponding author: E-mail: [email protected]. Tel.: +8605722023301
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Abstract
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Background: Saffron is one of the most expensive spices and is rich in pharmacological and biological active

ingredients.

Objective: We explored the community structure and diversity of the fungi growing on the surface of saffron corms

having different growth characteristics.

Methods: Thirty corm samples having different growth characteristics, i.e., infected, unable to germinate, able to

germinate but not bloom, able to germinate and produce one flower per corm, and able to germinate and produce two

flowers per corm, were collected from South Tai Lake Agricultural Park, Huzhou central hospital. The next-generation

sequencing (NGS) method was employed to detect the internal transcribed spacer (ITS) ribosomal RNA in fungus. The

community structure of fungi on the surface of corms having different growth characteristics was described using

bioinformatics tools, and the specific fungus on the corm surface was identified.

Results: The fungi on the surface of corms exhibited different developmental features. The abundance of

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Trichocomaceae and Talaromyces was high in infected corms, whereas that of Aspergillaceae was high in corms that

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were unable to germinate. Penicillium and Dothideomycetes were observed in corms that could germinate but not bloom,

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and in corms that could germinate and produce two flowers per corm, respectively. Differences were observed in

Cladosporium, Lambertella, Passalora, Penicillium, and Talaromyces among the five groups at the genus level.
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Conclusion: An understanding of the fungal diversity on the corm surface might provide useful information for
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improving germination and blooming of saffron corms.

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Keywords: Saffron, Corm, Fungi, Germinate, Bloom

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Introduction
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Crocus sativus L. is a perennial flower, belonging to the family Iridaceae, whose flowers are
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characterized by six petals, three stamens, and three red stigmas; saffron is the stigma of the C.

sativus flowers[1, 2]. Originally from Iran, saffron has been successfully grown in China, Spain, Italy,

India, Mexico, and other countries[3]. Saffron is considered one of the most expensive spices in the

world because of its unique way of growing and harvesting[4]. Saffron is used as not only a cosmetic

and coloring food additive but also as traditional medicine that is in use since ancient times[5, 6].
Crocetin glycoside, crocetin, and safranal are the three main pharmacological and biological active

ingredients of saffron[7], and recent in vitro and in vivo experiments have identified its antioxidant,

anti-tumor, anti-depression, anti-anxiety, anti-genotoxicity, anti-diabetes, antiplatelet,

anti-atherosclerosis, anti-inflammatory, and anti-convulsion properties, and other effects[7-14].

As C. sativus plants do not produce seeds, they are cultivated by corms, which are the

underground stems, making it a valuable crop. Fewer flowers per corm or unhealthy corms lead to a

lower quality and yield of saffron. Many factors such as soil, climate change, and microorganisms

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affect saffron cultivation and its yield[15-17], and increasing the number of flowers on per corm, while

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maintaining a healthy corm, could improve both quality and yield[18]. Corms are the organs for

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storage and vegetative reproduction; thus, they are no less important than roots[19]. Moreover,

previous studies have shown that mature and larger corms produce more flowers and subcorms[20].
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The microbial community within a plant’s interior, and on the leaves, stem, rhizosphere, and
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other parts significantly influences its growth during the entire growth cycle[21]. Previous studies have
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reported a complex relationship between the microorganisms from corms and environmental
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microorganisms, such as soil, rhizosphere, atmosphere, and container, and the growth and flowering

of corms[18, 20, 22-25]. Characters such as community structure and diversity of the bacteria in the
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rhizosphere, soil, corm, and leaves of saffron have been reported in many studies[18, 19, 23, 24, 26, 27]. In
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addition, the biological fertilizer and biological control characteristics of the bacteria isolated from

saffron have been studied[28]. Pathogenic, arbuscular mycorrhizal, and endophytic fungi have been

isolated from the saffron rhizosphere, soil, and corm, along with the active compounds of these

fungi[16, 23, 29, 30]. The dynamics of fungal diversity in the rhizosphere, corm layer, and massive soil of

saffron has also been studied[22].

 Even though we have previously observed significant differences in the species of fungi in

different soil samples, with no significant difference in the species and abundance of the main

bacteria, it remains unclear whether a similar difference exists in fungi from the surface of corms

having different growth characteristics. We, therefore, determined the internal transcribed spacer

(ITS) ribosomal RNA (rRNA) sequences of fungi to investigate the fungal communities and

diversity on the surface of corms with different growth characteristics.

Materials and methods

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Materials

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Samples were collected from the South Tai Lake Agricultural Park in Huzhou city and evaluated

by the research team at Huzhou central hospital, Huzhou, Zhejiang province. The fungi on the
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surface of saffron corms having different growth characteristics were investigated. After air drying,
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removing dust, and removing the outer layer, the appearance of the corms was observed. The fresh

weight of corms was, on average, 20.0 ±1.0 g, and the circumference and thickness were uniform.
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Corm annual production (kg/m2) was 2.42 ± 0.13. The pH of the soil was 6.95 ± 0.34. Humidity(%)
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of soil was 67.30 ± 4.32. Finally, 30 corms were collected and divided into five groups, each
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containing six corms. Group A, group B, group C, group D, and group E represented infected corms,
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corms that could not germinate, could germinate but not blossom, could germinate and produce one

flower per corm, and could germinate and produce two flowers per corm, respectively.

Methods

DNA extraction and PCR amplification

 We used the E.Z.N.A.® DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) to isolate DNA of the

fungi from the surface of corm samples, following the manufacturer’s protocols. The integrity of the

extracted genomic DNA was tested using 1% agarose gel electrophoresis. The ribosomal RNA gene

of fungal ITS was amplified using universal fungal primers. The forward and reverse primer

sequences were 5′- CTTGGTCATTTAGAGGAAGTAA-3′ and 5′-

GCTGCGTTCTTCATCGATGC-3′, respectively, and the PCR amplification procedure was

performed with an initial denaturation at 95°C for 2 min, followed by 95°C for 30 s, 55°C for 30 s,

t
repeated for 25 cycles, 72°C for 30 s and a final extension at 72°C for 5 min. For each sample, PCR

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amplification was repeated three times, and the PCR products of the same sample were mixed and

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detected by 2% agarose gel electrophoresis. The PCR products were recovered by AxyPrepDNA gel

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recovery kit (Axygen). After eluting with TrisHCl, the PCR products were detected by 2% agarose
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electrophoresis. According to the preliminary quantitative results of electrophoresis, the PCR

products were detected and quantified by QuantiFluor™-ST blue fluorescence quantitative system
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(Promega), and then mixed in proportion according to the sequencing requirements of each sample.
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Library Construction and Sequencing

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After purification, the samples were quantified using Qubit ®2.0 (Invitrogen, Carlsbad, CA,
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USA), and the amplification products of 24 bar codes were uniformly mixed. An Illumina
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double-terminal library was constructed using the pooled DNA products, and the amplicon library

was sequenced on an Illumina MiSeq platform (Shanghai BIOZERON Co., Ltd, China) following

the standard protocols. The sequences of all the strains were stored in the NCBI Sequence Read

Archive (SRA) database.

Sequencing data bioinformatics analysis

 Data Optimization and Statistics: First, according to the overlapping relationship between PE

reads, the pairwise reads were spliced into a sequence, and the quality of reads and the effect of

merge were filtered by quality control. The direction of the sequence was corrected according to the

box sequence at the end of the sequence, and the valid data were identified and distinguished

according to the barcode tag sequence.

Operational taxonomic unit (OTU) cluster analysis: The method of UPARSE (version 7.1) was

used to cluster OTU. The similarity of sequences in OTU was set to 97%, and the representative

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sequence of OTU was obtained. The chimeric sequences produced in PCR amplification were

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detected by UCHIME (version 4.2.40) and removed from OTU. The optimized map sequences were

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compared to OTU representative sequences by usearch global method, and the statistical tables of

sequence abundance of OTU samples were obtained.

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Diversity index analysis: The diversity of fungi was analyzed by Mothur
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software(http://www.mothur.org/wiki/Main_Page). We determined the coverage rate to reveal the

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index of sequencing depth. The Chao-the Chao1 estimator and Ace-the ACE estimator calculated the
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abundance index of fungi, and the index of fungal diversity was calculated by Shannon-the Shannon

index and Simpson-the Simpson index.

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Petal diagrams were used to visualize the number of OTUs that were exclusive or shared by

multiple samples in the present study.

Lefse (http://huttenhower.sph.harvard.edu/galaxy/root?tool_id=lefse_upload) conducted linear

discriminant analysis (LDA) on samples according to different grouping conditions and taxonomic

composition, to find out the communities or species that have significantly different influence on

sample division.
 The full link hierarchical clustering technique of R-packet HCLUST

(http://sekhon.berkeley.edu/stats/html/hclust.html) was used to cluster the genera obtained from RDP

classifiers. The distribution of fungi at the genus level was described by a histogram.

Statistical analysis

The statistical analysis of the data was conducted using SPSS 17.0. The data are represented as

mean ± standard deviation (SD). The differences of fungal richness and diversity among five groups

t
were statistically analyzed by the SNK test. The differences were considered statistically significant

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when a p-value < 0.05 was obtained.

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Results
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Fungal richness and diversity on the surface of corms with different growth characteristics
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As shown in Table 1, the coverage index indicated that the coverage of the genome library of

sequenced samples was greater than 99%. significant differences were observed in the Chao richness
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estimates among the five groups (p< 0.05); the lowest and highest richness estimates of fungi were in
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Groups A and D, respectively. The difference in Shannon index among the five groups was
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statistically significant (p< 0.05), and the lowest and the highest Shannon indices were found in

Groups A and D, respectively. These results revealed that the lowest richness and diversity of fungi
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were found in infected corms.

Fungal community structure on the surface of corms with different growth characteristics

Petal diagrams were used to count the number of common and unique OTU in multiple samples. As

shown in Figure 1, the petal diagram showed that shared OTUs were 65 and that there were 7, 4, 1,
25, and 8 unique OTUs in Groups A, B, C, D and E, respectively. These results indicate fewer

variations in fungal types among the five groups.

The cladogram in Figure 2 shows that there were significant differences in fungal abundance at

different taxonomic levels in different groups. Yellow circles represent species with no statistical

difference among the five groups. Red, green, blue, and purple circles, and azure green circles

represent the species that play an important role in Groups A, B, C, D and E, respectively. The

different species corresponding to lowercase letters are shown in the legends.

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Bar chart of LDA score distribution shows that the LDA value of significantly different species is

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greater than the preset value. The color of the column chart represents the respective group. The

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length of the bar chart represents the degree of influence of different species among different groups.
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As shown in Figure 3 and Table 2, there were 31 fungi with significant differences among the five

groups. The higher length in the Column chart indicates that some fungi such as Trichocomaceae,
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Talaromyces, Eurotiomycetes, Eurotiales, Aspergillaceae, Aspergillus, Cladosporiaceae,

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Capnodiales, Penicillium, Cladosporium, and Dothideomycetes had a more significant influence.

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The fungal community structure was further analyzed at the genus level. As shown in Figure 4,
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each column corresponds to the abundance of a species. The fungi with differences among the five
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groups are marked in red letters. The red, the earthen yellow, the green, the blue, and purple bar

charts show Groups A, B, C, D and E, respectively. The picture shows that Cladosporium,

Lambertella, Passalora, Penicillium, and Talaromyces had significant differences among the five

groups at the genus level.

Discussion
 In the past years, much research has been carried out on studying the relationship between saffron

and bacteria[18, 19, 26, 27], while relatively fewer researchers have studied saffron corm fungi. A

two-stage cultivation mode is used to cultivate saffron in China[3]. We investigated fungal diversity

on the differences of in saffron corm samples with different growth characteristics including infected,

could not germinate, could germinate but not bloom, could germinate and produce a flower per corm,

and could germinate and produce two flowers per corm.

Previously, on analyzing different soil samples, we observed no significant difference in the

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species and abundance of main bacteria, while a significant difference in the species of fungi was

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observed. We sought to understand whether a similar difference in the fungal species exists on the

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surface of corms, and the effect of these fungi on the growth and flowering in saffron. Corms with

different growth characteristics, such as, infected, unable to germinate, able to germinate but not
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bloom, able to germinate and produce one flower per corm, and able to germinate and produce two
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flowers per corm, therefore, were used as experimental materials, and the fungi on the surface of the
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corms were detected by the NGS technique. We observed differences in fungi on the surface of

corms having different growth characteristics, and our results suggest that the fungi on the surface of
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corms might affect the germination and flowering capacity of corms.

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Fungi constitute an immensely diverse population and form the core of ecosystem function. Fungi
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play a key role in energy and nutrient cycling and affects the composition of plant communities

through symbiosis[31]. They affect host plants mainly through the following two mechanisms: (1) the

production of different chemicals such as reactive oxygen species, plant toxins, plant hormones,

volatile organic compounds, poisons, antibiotics, and peptide phenols, (2) continuous perception and

recognition of host environments through light, chemical, and physical cues; they can also
alter/induce the overall gene expression of plant defense and metabolic pathways. This leads to the

secretion of effectors, enzymes, and secondary metabolites, altering the metabolism, and defense

against toxic compounds[29, 32, 33].

Numerous studies have reported that fungi, such as Rhizoctonia crocorum, Phoma crocoplila,

Fusarium moniliforme, Macrophomina phaseolina, Fusarium oxysporum, f. sp. solani, F.

pallidoroseum, F. equiseti,Mucor sp., Penicillium sp., Sclerotium rolfsii, Aspergillus, Cladosporium

cladosporoides, Rhizomucor sp., Paecilomyces sp., Phoma sp., Beauveria sp., Stromatinia,

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Cochliobolus, Rhizopus, Bipolaris spicifera, and Rhizopus nigricans, can affect the growth of saffron

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corm and cause corm diseases[16, 23, 34].

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The results of this study showed an abundance of Trichocomaceae and Talaromyces in infected
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corms, and differences were observed in Talaromyces among the five groups at the genus level. The

species belonging to Trichocomaceae are mainly saprophytic and represent some of the most
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destructive and non-metabolic microorganisms known[35]. They are related to food corruption, and
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mycotoxin production can occur in indoor environments and cause harm through the formation of

β-glucans, mycotoxins, and surface proteins. Some species are opportunity chlamydia, whereas
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others are used in biotechnology to produce enzymes, antibiotics, and other products[35]. Talaromyces
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belong to the Trichocomaceae family, and some species are isolated from soil, plants, sponges, and
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food[36]. Many studies have shown that Talaromyces have an antagonistic effect and biological

control potential against plant pathogens[37, 38].

Our results showed an abundance of Aspergillaceae in corms that were unable to germinate.

Aspergillaceae is characterized by the formation of bottle-shaped or cylindrical bottle-shaped vials[35].

The ascospores are produced in closed shell cells or surrounded by Hülle cells, mainly because the

ascospores have grooves or slits[35].

A difference in Cladosporium was observed among the five groups at the genus level.

Cladosporium is widely distributed, often isolated from soil, food, paint, textiles, and other organic

matter, or as a secondary intruder settled in the leaf lesions caused by plant pathogenic fungi[39].

Many Cladosporium species exist worldwide, and cause decay, deterioration, allergy, and even plant

or animal diseases, thereby having a significant impact on the environment[39]. As a biocontrol agent,

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Cladosporium sp.pl. control Phytophthora capsici by promoting hyphal deformation and are also

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used to control Botrytis cinerea on broad bean[40]. The metabolites of Cladosporium cladosporioides

display activity against plant pathogens as well[41].

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Penicillium was abundant on the surface of corms that could germinate but could not bloom,

and differences were observed in Penicillium among the five groups at the genus level. The main
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role of Penicillium in nature is to decompose organic matter, causing devastating decay. Penicillium
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sp.pl. act as pathogens for pre-harvest and post-harvest crops and produce a variety of mycotoxins[42].

Penicillium is a common fungal pathogen infecting saffron, causing corm decay[16].

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We observed an abundance of Lambertella in corms that could germinate but produced only one
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flower per corm, and there were differences in Lambertella among the five groups at the genus level.

Species of Lambertella mainly occur in leaves and fruits, and rarely in the fruiting bodies of roots,

branches, herbaceous stems, and other fungi. Most species of Lambertella are saprophytic, with a

few being herbivorous[43].

Our study also showed differences in Passalora among the five groups at the genus level. Three

kinds of Passalora related to the leaf spot of multi-flowered rose have been reported: Passalora
rosigena, Passalora rosae, and Passalora rosicola[44]. Leaf mildew caused by Passalora fulva is one

of the main diseases of tomato[45].

Further, we observed an abundance of Dothideomycetes, especially in corms that could germinate

and produce two flowers per corm. In a manner similar to that of saprophytic bacteria,

Dothideomycetes degrades plant biomass and plays a vital role in maintaining ecosystem and global

carbon cycle[46]. Dothideomycete is a macrofungus with high ecological diversity, including a variety

of plant pathogenic fungus species, and widely infects hosts[47]. One or more members of this

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category infect almost every major crop, including those that produce food, feed, fiber, and biofuels.

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In addition to accommodating important plant pathogens in vitro, this category also includes fungi

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with an unprecedented diversity of life history strategies and metabolic characteristics[47]. Secondary

metabolites are one of the earliest factors necessary for virulence and host specificity of necrotic
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bacteria in Dothideomycete[47].
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Taken together, our study showed significant differences in fungal species and their abundance on
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the surface of corms having different growth characteristics. This preliminary study provides certain
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guidance for preventing corm rot and further promoting normal germination and flowering. Small

sample size, single sample source area, and lack of clarity in the specific types of fungi were few of
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the limitations posed by our preliminary screening experiment. Further research, with expanded
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sample size, and including multiple areas for sample selection, would help in identifying specific

species of fungi on the surface of the corms and is essential to verify the results of our study.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of

the authors.
Authors’ contributions

All the authors participated in the conception and design of the study;

Conceived and drafted the manuscript: Da Miao and Shuwen Han;

Performed the experiments:Liu Jin;

Analyzed the data: Zhou Qing;

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Wrote the paper: Da Miao and Shuwen Han;

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All the authors read and approved the paper.

Competing Interests us
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The authors declare no conflicts of interest.
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Acknowledgments
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This work was supported by Zhejiang Medical and Health Technology Projects (No. 2020KY301).
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 Table 1. Fungal richness and diversity on corms with different growth conditions

group n OTU Chao Coverage Shannon Simpson

A 6 56.00±13.49 68.50±18.26 0.99906±0.00010 1.66±0.52 0.31±0.17

B 6 66.00±9.90 76.00±7.43 0.99930±0.00019 1.73±0.20 0.28±0.057

C 6 81.00±7.95 97.33±13.81 0.99900±0.00021 2.11±0.087 0.20±0.011

D 6 94.83±14.62 107.33±19.95 0.99889±0.00042 2.40±0.34 0.17±0.067

E 6 79.33±15.029 97.50±20.70 0.99889±0.00058 1.91±0.56 0.31±0.20

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F value — 8.519 5.691 — 3.563 1.636

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p value — p＜0.001 0.002 — 0.020 0.196

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Table 2. Each group showing a difference in the Fungal abundance on corms

group A group B group C group D group E

Eukaryota Eurotiomycet Geomyces Tremellomycetes Mycosphaerellace

Leotiomycetes es Eurotiales Cladosporiacea Filobasidiales ae Acremonium

Phialocephala Aspergillus e Cladosporium Plectosphaerellace Hypocreales

Helotiales Aspergillacea Capnodiales ae Sordariomycetes

Trichocomace e Penicillium Glomerellales Passalora

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ae Pseudeurotiace Rutstroemiaceae Didymellaceae

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Fung Talaromyces ae Lambertella Pleosporales

i Ascomycota

us Dothideomycetes
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The distinct fungi with different growth conditions from corms infectious, corms that could not

germinate, corms that could germinate but not bloom, corms that could germinate and bloom a
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flower each corm, and corms that could germinate and bloom two flowers each corm, determined
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using LDA Effect Size analysis are listed in the table. In each group, the fungi with a relatively
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higher abundance are marked in red.

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Figure 1. The number of OTUs of fungi on the surface of the corms

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Petal diagrams were used to count the number of common and unique OTU in multiple samples.
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The diagram shows the number of OTUs that were exclusive or shared in the 30 samples.
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Figure 2. The abundance of fungi on corms

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The cladogram shows significant differences in fungal abundance at different taxonomic levels in
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different groups, and the abundance of fungi on the surface of the corms in the 30 samples.
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Figure 3. Fungi on corms showing significant differences at the species level

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LDA Effect Size analysis was used to compare the differences in microbial abundance among the
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five groups. The figure shows 31 fungi with significant differences at the species level among the

five groups.
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Figure 4. Analysis of the fungi on corms at the genus level
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The fungi showing differences among five groups at the genus level are marked in red. The red,

earthen yellow, green, blue, and purple bar charts represent group A, group B, group C, group D, and
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group E, respectively. The results show that Cladosporium, Lambertella, Passalora, Penicillium, and
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Talaromyces had significant differences among the five groups at the genus level.
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