Molecular and Pathogenic Characterization of Fusarium Species Associated with Corm Rot Disease in Saffron from China

Saffron (Crocus sativus L.) is a commercial spice crop well-known throughout the world, valued for culinary, colorant, and pharmaceutical purposes. In China, Fusarium nirenbergiae was detected as causative agent of saffron corm rot, the most pervasive disease for the first time in 2020. In the present study, 261 Fusarium-like isolates were recovered from 120 rotted corms in four saffron producing fields at Zhejiang, Shanghai, and Yunnan provinces, China, in 2021. A combination of morpho-cultural features and multilocus sequence analysis (MLSA) of the concatenated rpb2 (DNA-directed RNA polymerase II largest subunit) and tef1 (translation elongation factor 1-α) partial sequences showed that the isolates from saffron belong to Fusarium nirenbergiae as well as F. commune, and F. annulatum with isolation frequencies of 58.2%, 26.8%, and 14.9%, respectively. Notably, F. commune was more prevalent than F. annulatum in the collected samples. Pathogenicity tests confirmed that both species were pathogenic on saffron corm. This is the first report of F. annulatum and F. commune causing corm rot of saffron, globally. Outcomes of the current research demonstrate that Fusarium spp. associated with saffron corm rot are more diverse than previously reported. Furthermore, some plants were infected by two or more Fusarium species. Our findings broaden knowledge about Fusarium spp. that inflict corm rot and assist the development of control measures.


Introduction
Crocus sativus (Iridaceae) is a bulbous perennial herb that is cultivated in warm temperate and subtropical countries throughout the world. Its vivid crimson stigmata and styles of the flowers are used as a desirable condiment, dye, aroma, antioxidant, and in medicine [1,2]. Saffron is a triploid, male-sterile plant incapable of developing fertile seeds for reproduction; this plant can be propagated only vegetatively by its corm [3]. The major saffron varieties include Aquilla, Crème, Kashmiri, Mongra, Organic, Persian, Spanish, and Superior [3][4][5]. Nevertheless, only the "Fanhong Hua1" cultivar is widely cultivated in China. This plant is widely used as an important herbal medicine crop in China with a total cultivating area of nearly 600 hectares.
Fusarium is a ubiquitous genus of filamentous fungi with varying morphological, physiological, and ecological features that causes economic damage on agricultural products and includes opportunistic human pathogens [11,12]. However, there are non-pathogenic strains of Fusarium that are used in plant protection [12]. In addition, these fungi are able to synthesize phytotoxins and mycotoxins as secondary metabolites, which can play an important role in the pathogenesis [13,14]. To date, more than 400 phylogenetically distinct species in 23 monophyletic species complexes are included in the genus Fusarium. Several monotypic lineages have been characterized of which members of the F. solani species complex (FSSC), F. oxysporum species complex (FOSC), F. fujikuroi species complex (FFSC), F. incarnatum-equiseti species complex (FIESC), and F. sambucinum species complex (FSSC) cause considerable diseases in plants [11,12,[15][16][17][18][19]. Some Fusarium species are considered to be hemibiotrophs capable of switching to necrotrophs depending on the host and environmental conditions [14,20]. Furthermore, it has been difficult to distinguish closely related Fusarium species, due to inter-/intra-species overlap and inconsistency in morphological traits. By applying a polyphasic taxonomic approach that combines morphological observations with DNA fingerprinting, and multilocus phylogenetic analysis (MLSA), numerous species have been delineated within Fusarium spp. complexes, leading to significant improvement in the Fusarium classification system [11][12][13]19,21,22].
Limited information is available on the genetic diversity, phylogenetic relationships, and epidemiology of Fusarium species causing saffron corm rot in China and elsewhere in the world. Therefore, the aim of the current research was to identify and characterize Fusarium spp. associated with the disease on Crocus sativus through pathogenicity tests, morphological data, and molecular methods.

Fungal Isolation and Morphological Characterization
Saffron plants (120) with rotted corms were sampled from four saffron cultivation areas in Zhejiang (Jiande and Wuyi cities), Yunnan (Shangri-la city), and Shanghai (Chong Ming Dao Island) provinces, China (Table 1). Excised symptomatic tissues consisting of diseased and healthy parts were surface-sterilized with a 2% solution of sodium hypochlorite (0.1% active ingredient of chlorine) for 1 min and 75% ethanol for 30 s. The samples were then washed thrice with sterile distilled water, air-dried on the sterile filter papers under aseptic conditions, and finally plated onto Potato Dextrose Agar (PDA) plates, which were incubated in the dark at 25 • C. Purified isolates were obtained by hyphal tipping; then, they were sub-cultured on PDA and synthetic nutrient-poor agar (SNA) media [11,23]. Morphological characteristics of fungal colonies were meticulously examined under a Nikon Eclipse microscope (Tokyo, Japan).

DNA Sequencing and Molecular Phylogenetic Analysis
The mycelium of 7-day-old isolates was harvested from PDA by scraping the colony surface, freezing the mycelium in liquid nitrogen, and then grounding with a sterile mortar and pestle. The genomic DNA of thirteen representative isolates was extracted using a Plant Genomic DNA kit (Tiangen, China) according to the company's protocols. Portions of nuclear translation elongation factor 1-alpha (tef1), second largest subunit of RNA polymerase II gene (rpb2), and internal transcribed spacer (ITS) genes were amplified from the thirteen representative isolates using the primers EF-1/EF-2, RPB2-5f2/RPB2-7cr, and ITS1/ITS4, respectively [10,11]. Polymerase chain reaction (PCR) was conducted with 1 µL forward primer (10 µM), 1 µL reverse primer (10 µM), 0.5 µL DNTPs (10 mM), 12.5 µL of 2× Rapid Taq Master Mix (Vazyme, Nanjing, China), 1 µL of genomic DNA, and 9.5 µL of DNase-free water. The PCR program consisted of an initial denaturation at 95 • C for 2 min, 35 cycles of denaturation at 95 • C for 30 s, annealing for 30 s at 55 • C, and an extension at 72 • C for 2 min, followed by a final extension (72 • C, 10 min). The amplified products were visualized on a 1% agarose gel and then sequenced in both directions to ensure high accuracy by Sangon Biotech Co., Ltd. (Shanghai, China). All sequences obtained in this study were deposited in GenBank and the accession numbers are included in Table 2. We performed BLASTN searches via the NCBI BLAST web portal (available online: https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 15 August 2021) to gather related sequences for inclusion in phylogenetic analysis. BLASTN searches were performed against the Nucleotide collection (nr/nt) and included searches were restricted to sequences from type material. After finalizing the multiple alignments by MUSCLE [24] for individual and concatenated loci (rpb2 + tef1), the Kimura two-parameter model assuming a discrete gamma distribution and invariant sites (K2 + G + I) was used to estimate the best substitution models. Phylogenetic inference was obtained using maximum likelihood (ML) in MEGA X (Molecular Evolutionary Genetics Analysis version 10.2.4 [25]. Branch stability was estimated with 1000 bootstrap replicates. We included sequences from type strains of Fusarium species initially identified as closely related to our sequences (Table 3) by preliminary BLAST searches.

Pathogenicity Studies
An in vitro virulence test was conducted to evaluate the ability of thirteen representative isolates of Fusarium species to colonize saffron and induce rot symptoms with two methods. In the first method, inoculation of corms was done as described by Palmero et al. [7] with slight modifications. Briefly, the intact bulbs were surface-disinfested with 5% sodium hypochlorite solution (0.26% active ingredient of chlorine) for 10 min followed by 75% ethanol for 1 min and then rinsed three times in sterile water. Each isolate was cultured on PDB (potato dextrose broth) medium for 7 days at 25 • C under shaking (150 rpm) conditions. Conidial suspensions were filtered through three layers of sterilized gauze and centrifuged at 8000 rpm for 10 min. To remove the PDB, the conidial pellet was washed three times in sterile distilled water. The spores were resuspended to a final concentration of 1 × 10 6 conidia/mL for inoculation. To completely absorb pathogens, the corms were submerged in the spore suspension and then planted in an aseptic substrate (black/white peat, perlite, vermiculite; 2:1:1) and maintained for 21 days under controlled conditions in a growth chamber with 12 h photoperiod, 23 ± 2 • C, and 70% relative humidity. The controls were inoculated with sterile water. In the second method, the mycelial plugs (5 mm diameter) were placed onto sterilized bulbs and wrapped with Parafilm, then incubated in the same condition as mentioned above for 14 days. Non-colonized PDA discs were used as negative controls. The experiments were repeated twice. The disease progression on inoculated plants was inspected daily for up to three weeks and visual observations recorded. Koch's postulates were fulfilled by reisolating and identifying the fungal isolates from symptomatic corms.

Field Survey, Disease Symptoms, and Pathogen Isolations
In 2021, a total of 120 diseased Crocus sativus plants exhibiting corm rot, leaf chlorosis, and wilted shoots were collected from four saffron-growing areas in China (Figure 1). Overall, 261 Fusarium-like isolates were obtained from symptomatic corms tissues (Table 1). Most isolates were obtained from individual rotted area on the corms. Based on their colony characteristics as well as molecular methods, the isolated fungi were identified as three species of Fusarium, namely F. annulatum, F. commune, and F. nirenbergiae. The isolation frequency of F. nirenbergiae (58.2%) was greater than F. commune (26.8%), and F. annulatum (14.9%). The ability of F. nirenbergiae to cause corm rot has been previously established [10], and was therefore not further examined in the current study. Here, we focused on thirteen representative isolates of F. annulatum, and F. commune, chosen based on geographical regions and identified using sequences of tef1 and ITS (internal transcribed spacer). The presence of one, two, and occasionally three diverse Fusarium species was confirmed from some samples based on the tef1 and ITS sequences.

Morphological Identification
Morphological features of the thirteen representative isolates recovered from symptomatic corms were consistent with the morphological descriptions of F. annulatum and F. commune [19,26]. On PDA, the abundant aerial mycelium of F. annulatum was pinkish white to maroon. The macroconidia were fusiform, cylindrical, narrow, straight to slightly curved with 3-6 septa. No chlamydospores were found. The microconidia were typically aseptate, club-shaped each with curved apical cell; these developed on monophialides and polyphialides (Figure 2). On PDA, Fusarium commune formed densely

Morphological Identification
Morphological features of the thirteen representative isolates recovered from symptomatic corms were consistent with the morphological descriptions of F. annulatum and F. commune [19,26]. On PDA, the abundant aerial mycelium of F. annulatum was pinkish white to maroon. The macroconidia were fusiform, cylindrical, narrow, straight to slightly curved with 3-6 septa. No chlamydospores were found. The microconidia were typically aseptate, club-shaped each with curved apical cell; these developed on monophialides and polyphialides (Figure 2). On PDA, Fusarium commune formed densely floccose to fluffy aerial mycelium with white to pale lilac colony. The macroconidia were banana-shaped, with 3-5 septa, forming sporodochia. The microconidia were cylindrical to ovate-oblong, primarily aseptate, and nesting on aerial polyphialides or, less often, on monophialides. Spherical, intercalary, or terminal chlamydospores were produced singly or in pairs (Figure 2).

Morphological Identification
Morphological features of the thirteen representative isolates recovered from symptomatic corms were consistent with the morphological descriptions of F. annulatum and F. commune [19,26]. On PDA, the abundant aerial mycelium of F. annulatum was pinkish white to maroon. The macroconidia were fusiform, cylindrical, narrow, straight to slightly curved with 3-6 septa. No chlamydospores were found. The microconidia were typically aseptate, club-shaped each with curved apical cell; these developed on monophialides and polyphialides (Figure 2). On PDA, Fusarium commune formed densely floccose to fluffy aerial mycelium with white to pale lilac colony. The macroconidia were banana-shaped, with 3-5 septa, forming sporodochia. The microconidia were cylindrical to ovate-oblong, primarily aseptate, and nesting on aerial polyphialides or, less often, on monophialides. Spherical, intercalary, or terminal chlamydospores were produced singly or in pairs (Figure 2).

Molecular Characterization and Phylogeny
The PCR amplification of tef1, ITS, and rpb2 regions for isolates from saffron generated 647, 542, and 941 bp fragments, respectively. BLASTN of ITS sequences indicated close similarity with Fusarium species but provided insufficient resolution to identify the species. On the other hand, the tef1 and rpb2 indicated that five of the isolates were closely related to the type strain of F. annulatum while the other eight isolates were closely related to F. commune, which supported our preliminary morphological identification of these isolates. Isolates WFA10, WFA18, JFA12, JFA15, and SFA4 showed close sequence similarity with F. annulatum at both the tef1 and rpb2 loci. These isolates shared 99.05% identity with F. annulatum type strain CBS258.54 [19,27] at the tef1 locus and 99.37% to this type-strain at the rpb2 locus. Isolates YFC2, YFC5, JFC1, JFC7, SFC6, SFC20, WFC3, and WFC8 shared 97.45% identity with the type strain of F. commune CBS 110090 [11] at the rpb2 locus. No tef1 sequence is available for the F. commune type strain; however, the tef1 of the four isolates shared 100% identity with tef1 sequences of other strains designated as F. commune (e.g., GenBank accessions MG888467.1, MF150040.1, MW589548.1, MT313846.1). To further elucidate and illustrate the phylogenetic relations, we generated phylogenetic trees based on tef1 and rpb2, including sequences of isolates recovered from saffron, Fusarium type strains plus non-type strains of F. commune. These trees further supported the identification of eight isolates as F. commune and five isolates as F. annulatum (Supplementary Figures S1 and  S2). The phylogenetic tree, based on the concatenation of two genes (rpb2 + tef1) spanning 1476 nucleotides among 80 ingroup strains, included three main clades corresponding to FOSC, FFSC, and F. commune. The MLSA tree illustrated that isolates collected from saffron in the present study clustered strongly with F. annulatum [19] and F. commune type strains [26,28] with bootstrap values 95% and 100%, respectively (Figure 3). The topology of the multilocus tree was similar to the phylogenetic trees constructed from the individual genes ( Supplementary Figures S1 and S2). Moreover, F. nirenbergiae JD1, which is a major causative pathogen of saffron rot in China, also falls unambiguously within the F. nirenbergiae clade (88% bootstrap value), which belongs to FOSC (Figure 3).

Pathogenicity Assays
Conidial and mycelial inoculation with thirteen representative isolates resulted in rotting and wilting symptoms in corms three weeks after inoculation under laboratory conditions. These symptoms were similar to field observations, whereas no symptoms

Pathogenicity Assays
Conidial and mycelial inoculation with thirteen representative isolates resulted in rotting and wilting symptoms in corms three weeks after inoculation under laboratory conditions. These symptoms were similar to field observations, whereas no symptoms were observed on control plants. The visual assessments indicated that the disease development in the corms inoculated by mycelial plugs was faster than conidial inoculation. The fungal isolates were reisolated from the inoculated corms and identified using tef1 locus to fulfill Koch's postulates ( Figure 4). As such, it was verified that F. annulatum and F. commune were capable of causing rot in corms. While the virulence of F. commune isolates was visually greater than F. annulatum isolates, the corm rot symptoms incited by each species were not distinguishable. were observed on control plants. The visual assessments indicated that the disease development in the corms inoculated by mycelial plugs was faster than conidial inoculation. The fungal isolates were reisolated from the inoculated corms and identified using tef1 locus to fulfill Koch's postulates ( Figure 4). As such, it was verified that F. annulatum and F. commune were capable of causing rot in corms. While the virulence of F. commune isolates was visually greater than F. annulatum isolates, the corm rot symptoms incited by each species were not distinguishable.

Discussion
Saffron corm rot is the most challenging disease of saffron with a high incidence in the Chinese provinces Shanghai, Yunnan, and Zhejiang. Fusarium species are among the most severe pathogens that affect a broad range of crops worldwide. We previously established that F. nirenbergiae is a causative agent of corm rot on saffron [10]. In the current study, we isolated and identified two further species from saffron growing regions, namely Fusarium annulatum and F. commune, based on morphological criteria and

Discussion
Saffron corm rot is the most challenging disease of saffron with a high incidence in the Chinese provinces Shanghai, Yunnan, and Zhejiang. Fusarium species are among the most severe pathogens that affect a broad range of crops worldwide. We previously established that F. nirenbergiae is a causative agent of corm rot on saffron [10]. In the current study, we isolated and identified two further species from saffron growing regions, namely Fusarium annulatum and F. commune, based on morphological criteria and multilocus (two-gene) phylogenetic analyses. Isolates identified as these two species were clearly distinct from F. nirenbergiae isolates previously described [10]. The establishment of Koch's postulates indicated that F. annulatum and F. commune isolates were pathogenic to saffron. However, they have a slight variation in virulence. The occurrence of these pathogens in different locations of China suggests that infected corms may serve as a source of inoculum for C. sativus infection.
Fusarium commune is associated with wilt and root rot diseases in a range of crops: Acacia koa, barley, carnation, carrot, Chinese water chestnut (Eleocharis dulcis), Douglas-fir, horseradish, maize, peas, rice, soybean, sugarcane, tobacco, tomato, and white pine. This fungus was originally misidentified as F. oxysporum; however, it has been resolved within the FOSC and described as a distinct species since 2003 [28][29][30][31][32]. Fusarium annulatum is a morphologically and phylogenetically diverse species which has been recently demonstrated as distinct from F. proliferatum [19]. F. annulatum is a member of the FFSC, which has been recorded as a pathogen on more than 200 plant hosts primarily in subtropical countries [11,19,33,34].
The rpb2 and tef1 genes possess high discriminatory power and are well represented in the GenBank database. The tef1 locus is frequently used as the first choice for taxonomic studies of Fusarium due to its single-copy occurrence and high degree of sequence polymorphism among closely related species, while rpb2 is the second-best gene for discriminating between closely related species (Supplementary Figures S1 and S2) [11,13,16]. In the combined rpb2 + tef1 tree, the saffron pathogens were resolved into the three species F. annulatum, F. commune, and F. nirenbergiae with high support values. It is worth mentioning that the ITS data were not used in the MLSA, due to their excessive variability within Fusarium [10,11,35] and their inability to resolve species.
The fungi isolated in this study from saffron morphologically resembled F. oxysporum; however, on close examination, they could be discriminated from each other as F. annulatum and F. commune based on morphological criteria. Although both species form polyphialides, F. commune also produces long, slender monophialides; these microscopic features distinguish F. annulatum and F. commune from F nirenbergiae and F. oxysporum [11,13,23,26,28]. Chlamydospores were only absent in F. annulatum [19]. The morphological identification validity was corroborated by phylogenetic analysis derived from the molecular data.
In spite of our efforts, we have not yet been successful in establishing species-specific diagnostic features for F. annulatum, F. commune, and F. nirenbergiae which induced rot on saffron plants. Further studies are needed to fully determine the pathogen-specific symptoms of corm rot. Additionally, some rotted corms exhibited slightly different symptoms in terms of severity, intensity, color, or shape, possibly due to secondary infection by saprobic bacteria and fungi or environmental conditions such as humidity or mechanical injury, as has been documented in previous studies [30,35,[38][39][40][41].
Strains of F. annulatum and F. commune are reported as pathogens of bakanae and root rot diseases in rice, respectively [19,28,[42][43][44]. Saffron is commonly planted after rice in China as a rotation. This raises the intriguing possibility that rice might serve as a reservoir or alternative host for pathogens of saffron and/or vice versa [19,28,[42][43][44]. A first step in investigating that hypothesis will be to investigate the host ranges of these strains: are they able to colonize and/or infect both rice and saffron?

Conclusions
Overall, data obtained in this study confirm Fusarium species are a serious limitation for the commercial production of saffron. Although F. nirenbergiae was the prevalent species inciting corm rot in the surveyed areas, F. annulatum, and F. commune were also recovered from diseased plants, showing to be very aggressive and virulent on saffron. In a disease complex, the frequency of pathogens may vary due to cultivars, agricultural practices, meteorological and climatological parameters, etc. In addition, our survey provides an overview on the biodiversity, distribution, and etiology of Fusarium spp. associated with corm rot of C. sativus in China, thus enabling the development of better environmentally friendly management strategies.

Supplementary Materials:
The following supporting information can be downloaded at: 'https: //www.mdpi.com/article/10.3390/jof8050515/s1, Figure S1: Phylogeny of Fusarium isolates from saffron, based on the partial rpb2 gene. Sequences from type strains are indicated by a circle (•). Sequences from strains isolated from saffron are indicated by a star ( ). Clades including saffron isolates are shaded in color. Evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model [45]. The tree with the highest log likelihood (-3483.33) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 84 nucleotide sequences. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 648 positions in the final dataset. Evolutionary analyses were conducted in MEGA X version 10.2.4 [25,46] and the tree image was generated using the Interactive Tree Of Life (iTOL) v5 [47]; Figure S2: Phylogeny of Fusarium isolates from saffron, based on the partial tef1 gene. Sequences from type strains are indicated by a circle (•). Sequences from strains isolated from saffron are indicated by a star ( ). Clades including saffron isolates are shaded in color. Evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model [45]. The tree with the highest log likelihood (-3460.04) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 87 nucleotide sequences. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 583 positions in the final dataset. Evolutionary analyses were conducted in MEGA X version 10.2.4 [25,46] and the tree image was generated using the Interactive Tree Of Life (iTOL) v5 [47]. References [45][46][47] are cited in the supplementary materials.   Table 2). The data presented in this study are openly available in NCBI. Publicly available datasets were analyzed in this study. These data can be found here: https://www.ncbi.nlm.nih.gov/ (accessed on 15 August 2021).

Conflicts of Interest:
The authors declare no conflict of interest.