New Insights into the Diversity and Pathogenicity of Fusarium Species Causing Carnation Wilt in Lam Dong, Vietnam

doi:10.21203/rs.3.rs-6859853/v1

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New Insights into the Diversity and Pathogenicity of Fusarium Species Causing Carnation Wilt in Lam Dong, Vietnam

https://doi.org/10.21203/rs.3.rs-6859853/v1

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published 06 Aug, 2025

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Carnation Fusarium wilt (CFW) is a devastating systemic disease affecting carnation production worldwide, yet little is known about its status in Vietnam. This study provides the first comprehensive report on the population and pathogenesis profile of Fusarium species associated with CFW in Vietnam. A survey of commercial greenhouses revealed approximately 10.7% disease incidence, with symptomatic plants showing chlorosis, wilting, leaf blight, and vascular discoloration. Twenty-one Fusarium isolates were recovered and classified into the Fusarium oxysporum species complex (FOSC) and F. incarnatum-equiseti species complex (FIESC), each comprising two morphologically distinct clades. Molecular identification based on ITS and TEF-1α sequences confirmed four species: F. oxysporum (9 isolates), F. pernambucanum (7), F. sulawesiense (4), and F. nirenbergiae (1), revealing notable species diversity. Pathogenicity tests at the seed germination and transplant stages demonstrated that all species were capable of causing disease, with varying degrees of aggressiveness. FOSC isolates generally delayed germination, while FIESC isolates caused a more significant reduction in germination rate. Both complexes induced root rot, seedling death, and foliar symptoms in transplants. Two highly virulent isolates, C1611 (F. oxysporum) and C2111 (F. pernambucanum), significantly reduced seed viability and survival and caused high disease intensity. Some isolates showed organ- or stage-specific pathogenicity. This is the first report of F. pernambucanum, F. sulawesiense, and F. nirenbergiae associated with carnation, globally, and the first record of CFW-associated species in Vietnam. These findings highlight the diversity and pathogenic complexity of Fusarium species involved in CFW and underscore the need for accurate identification and effective disease management strategies in local and international trade.

population

pathogenicity

Fusarium wilt

carnation

Vietnam

Carnation (Dianthus caryophyllus), a member of the family Caryophyllaceae, is among the most widely cultivated ornamental plants globally. Valued for its vibrant colors, long postharvest longevity and aesthetic appeal, carnation ranks as the second-most traded cut flower worldwide, holding a significant position in the global floriculture industry (Filgueira-Duarte et al., 2024). Native to the Mediterranean region, it has been successfully introduced to Vietnam, particularly well adapted to the cool-climate regions such as Lam Dong Province.

With agricultural intensification and mounting impacts from climate change, infectious plant diseases have become increasingly critical factors affecting crop productivity and economic sustainability, including for carnations. Among the most devastating diseases afflicting carnation is Fusarium wilt (FW), a vascular wilt disease caused primarily by members of the Fusarium oxysporum species complex (FOSC). Initially referred to as “vascular wilt,” or “crown rot” (Bijl, 1916), this disease causes yellowing of leaves, stunting, wilting, vascular discoloration (browning), cortical root rot, and necrosis progressing acropetally, ultimately resulting in plant collapse and death (Brayford, 1996; Garibaldi & Gullino, 1987; Basallote-Ureba et al., 2016).

Fusarium spp. are soilborne pathogens that infect plants through the roots and colonize the xylem vessels. They exhibit a biotrophic phase initially, which later transitions to a necrotrophic phase, occluding vascular tissues through mycelial proliferation, disrupting water and nutrient uptake and eventually killing the plant (Pérez Mora et al., 2024). Disease intensity varied by region depending on host genotype, environmental factors, cropping systems, and pathogen virulence (Horst & Nelson, 1968; Bijl, 1916; Robinson, 1961). In susceptible cultivars, yield losses may exceed 70% in severely infested fields (Katoch, 1999; Prados-Ligero et al., 2007). Even under protected conditions, FW incidence rates of 20–45% have been reported (Dhinesh et al., 2014).

The primary etiological agent of FW in carnation is F. oxysporum f. sp. dianthi, a forma specialis within the FOSC, with up to 11 races identified globally (Chiocchetti et al., 1999), race 2 being the most widespread (Vergara et al., 2007; Castano et al., 2014). However, mounting evidence indicates that the Fusarium population associated with carnation is taxonomically and phylogenetically heterogeneous. Studies from different geographical regions have implicated multiple Fusarium species (e.g., F. avenaceum, F. graminearum, F. proliferatum, F. culmorum, F. solani, F. tricinctum, F. globosum, F. incarnatum, and F. equiseti), in association with carnation wilt, occasionally co-infecting or exhibiting overlapping symptom profiles (Wright et al., 1997; J. Zhang et al., 2013; López et al., 2014; Shahbazi et al., 2021a; Cer & Benlioglu, 2024). These findings highlight the complexity of the pathogen composition and emphasize the importance of regional pathogen profiling.

Intra- and interspecific variability in virulence, reproductive strategies, and secondary metabolite production among Fusarium spp. further complicate disease diagnosis and management. Accurate identification of causal agents is therefore critical and typically involves an integrative approach combining morphological and molecular diagnostics. Although morphological features (e.g. colony pigmentation, conidial morphology, chlamydospore presence, …) can assist in species complex-level identification, phenotypical variability often limits taxonomic resolution. Consequently, molecular techniques have become indispensable. The translation elongation factor 1-alpha (TEF-1α) gene is widely used for species-level discrimination within Fusarium, due to its superior phylogenetic informativeness compared to the internal transcribed spacer (ITS) region, which often lacks resolution among closely related taxa (O'Donnell et al., 2009; O’Donnell et al., 2022). A polyphasic identification approach combining TEF-1α sequencing with morphological and pathogenicity assays provides a more robust framework for pathogen characterization.

Control of Fusarium wilt remains challenging due to the pathogen’s ability to persist in the soil as dormant propagules (chlamydospores) for extended periods (Roberts et al., 2024). The elimination or long-term suppression of soilborne inoculum is hindered by the absence of effective chemical treatments and limited host resistance (Roberts et al., 2024; Sharma et al., 2017). While benzimidazole fungicides such as carbendazim and benomyl were once effective (Ebben, 1977; Singh et al., 2016), their use has been banned in many countries, including Vietnam, due to regulatory, environmental and health concerns. Moreover, newly developed fungicides generally offer limited systemic protection against vascular-colonizing pathogens, reinforcing the need for alternative strategies such as soil disinfestation, biological control, and disease forecasting.

In Vietnam, carnation cultivation remains scattered but holds increasing economic relevance, particularly in Lam Dong province, renowned for its temperate climate and floriculture exports. Despite the long-recognized occurrence of FW, studies on the diversity of its causal agents remain scarce. The disease is frequently attributed to F. oxysporum f. sp. dianthi, yet emerging evidence from other crops and regions suggests the possible involvement of multiple Fusarium spp. in pathogenesis. Observations of disease resurgence in Lam Dong, often with heightened severity and recalcitrance to control, raise concerns over evolving pathogen populations potentially exacerbated by climatic stressors and monoculture practices.

Currently, little is known about the species composition and virulence patterns of Fusarium spp. affecting carnations in Lam Dong. Given the pathogen’s soilborne nature and the ecological implications of chemical control (Dusengemungu, 2021), comprehensive identification, including morphological assessment and virulence testing, is vital for developing targeted, efficient, and sustainable management strategies.

The present study aims to elucidate the species composition and pathogenic variability of Fusarium spp. associated with carnation Fusarium wilt in Lam Dong Province, Vietnam. By employing a combination of morphological diagnostics, molecular sequencing, and pathogenicity assays, we seek to improve understanding of the etiological complexity underlying CFW in this key production area, thereby enabling the development of locally adapted and sustainable management strategies, and supporting regional and international phytosanitary efforts.

2.1. Survey, sampling and isolation of pathogens

A survey was conducted in nine commercial carnation greenhouses in Da Lat and nearby areas (Lam Dong Province, Vietnam) to assess the occurrence of Fusarium wilt (Table 1). In each greenhouse, disease incidence was evaluated at five diagonal 1 m² plots. Incidence was calculated as the percentage of symptomatic plants among the total observed in each plot, and the average incidence per greenhouse was determined.

From each greenhouse, three symptomatic plants were randomly collected, wrapped in two layers of paper, and transported to the Plant Disease Laboratory, Dalat University. The causal agent was isolated from symptomatic stems following standard procedures. Stems were washed with soap and tap water, surface-disinfested in 0.1% sodium hypochlorite for 2 min, then in 70% ethanol for 2 min, followed by three rinses with sterile distilled water. Three transverse stem sections containing both healthy and diseased tissue were placed on half-strength Potato Dextrose Agar (PDA; Hi-Media, India) and incubated in the dark at 25°C for 3–5 days. Fusarium-like colonies were sub-cultured on fresh PDA, and pure cultures were obtained using the single-spore technique.

2.2. Morphological Characterization

Cultural characteristics and growth rates were assessed on PDA, while microscopic features were examined on carnation leaf agar (CLA) medium (Leslie & Summerell, 2006). A 0.5 cm disc from the edge of a 7-day-old PDA culture was placed at the center of fresh PDA plates and incubated at 25 ± 1°C in the dark. Each isolate was tested in triplicate. Macroscopic traits such as pigmentation, colony shape, margins, and growth pattern, were recorded. Colony diameter was measured every two days until the colony reached the plate edge, and radial growth rate (cm/day) was calculated.

Microscopic features were assessed on CLA medium prepared according to Fisher et al. (1982): fresh carnation leaves were cut into 1–2 cm pieces, autoclaved, and placed (3–5 pieces per plate) on 2% Water Agar. A 0.5 cm disc from each isolate was transferred to the CLA plates and incubated at 25°C in the dark. After 2–3 weeks, microconidia, macroconidia, and chlamydospores were examined under a compound microscope using slide preparations, with at least 30 structures measured per isolate.

Fusarium species complexes were initially identified by comparing cultural and microscopic traits with taxonomic keys (Leslie & Summerell, 2006; Lombard et al., 2019) and online resources such as FusaHelp.com. Following the preliminary identification of Fusarium isolates, a morphology-based hierarchical clustering analysis was conducted to further assess inter-isolate similarity patterns. The analysis utilized a dataset comprising 41 morphological attributes, including 35 binary-coded variables and 6 numerical variables describing colony pigmentation, mycelial growth pattern, and the presence, shape, size, and septation of macroconidia and microconidia, as well as chlamydospores, conidiophores, phialides, and sporodochia, resulting in a dendrogram of morphologically distinct groups (Table S1).

2.3. Molecular Identification

2.3.1 Genomic DNA extraction and polymerase chain reaction (PCR)

Molecular identification was performed for seven representative isolates of each phenotypic group. Fungal genomic DNA was extracted from approximately 100 mg of 7-day-old mycelium grown on PDA using a modified CTAB method. DNA concentration and purity were measured using a NanoDrop Microvolume Spectrophotometers (Thermo Scientific). Polymerase chain reactions (PCRs) were carried out to partially amplify the ITS region of the rRNA and TEF-1α genes using the universal primer pair ITS4/ITS5 (White et al., 1990) and EF1/EF2 (O’Donnell et al., 1998). The 25 µL reaction volume included 12.5 µL master mix (Bioline, USA), 1 µL of each primer, 2 µL of DNA template and molecular water. The thermal cycle was: initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 52°C (for ITS) or 54°C (TEF-1α) for 30 s, and 72°C for 30 s, with a final extension at 72°C for 5 min. PCR amplicons were checked by agarose electrophoresis (1.5%, at 100 V) using TBE buffer, purified using the TopPURE® PCR/GEL DNA PURIFICATION KIT (ABT, Ha Noi, Vietnam), and sequenced by the Sanger method (DNAsequencing, Can Tho, Vietnam).

2.3.2 Phylogeny analysis

The obtained sequence data were used for species identification by conducting BLASTn searches against the Fusarium-ID database and GenBank. Based on these comparisons, appropriate reference sequences were retrieved from GenBank (Table S2) using BioEdit v7.2. Multi-sequence alignments of concatenated ITS and TEF-1α regions from representative isolates and 60 GenBank references were performed using MEGA11 (Tamura et al., 2021) with ClustalW (https://www.genome.jp/tools-bin/clustalw). The best-fit model (TIM2e + G4) was selected based on Bayesian Information Criterion (BIC) criteria, and a Maximum Likelihood (ML) phylogenetic tree was constructed with 1,000 bootstrap replicates using IQ-TREE v4.2 (Nguyen et al., 2015). The resulting tree was visualized and edited using FigTree v4 and Adobe Illustrator CS6 (Adobe Inc., 2019). Sequences of the identified Fusarium isolates were deposited in GenBank, and accession numbers were assigned (PQ804582-PQ804588).

2.5. Pathogenesis tests

The pathogenicity of Fusarium spp. isolates was evaluated on carnation plants (cv. Samon) at both germination (blotter assay) and transplant (cutting assay) stages. Seeds and transplants were inoculated by immersion in a spore suspension, followed by sowing or potting. Effects on seed germination, seedling development, and transplant growth were recorded. Spore suspensions were prepared by adding 5 mL of sterile distilled water (0.1% Tween 20) to 15-day-old cultures (confirmed sporulation). Spores were released using a glass rod, filtered through sterile cotton, and adjusted to 10⁷ spores/mL using a Neubauer hemocytometer (Germany).

2.5.1 Seed assay

All Fusarium isolates were tested at the germination stage. Carnation seeds were soaked and shaken (150 rpm) in the spore suspension for 1 hour, then dried on absorbent paper for 1 hour. Ten seeds were placed in each plastic cup (6 × 6 × 4 cm) on moist filter paper (2 mL sterile water). Control seeds were treated with sterile distilled water containing 0.1% Tween 20. Ten replicate cups were used per treatment. Cups were covered and incubated in the dark at 25°C for 1 day, then exposed to a 12-hour artificial photoperiod. Sterile tap water (2 mL) was added every 2 days. Germination rate (%) and seedling survival (%) were recorded at 4, and 8 days post-inoculation (dpi). Pathogenicity was evaluated based on effects on germination, seedling viability, and seedling vigor. Vigor index (VI) was calculated following Dezfuli et al. (2008):

VI = Germination (%) × Total seedling length (cm).

2.5.2 Cutting assays

Seven Fusarium strains representing identified species were selected for transplant assays. Twenty-day-old cuttings from disease-free nurseries were used. Roots were trimmed to 1 cm and plants were immersed in a 10⁷ spores/mL suspension for 1 hour. Inoculated plants were potted in sterilized coconut fiber substrate (Coco, Finom, Vietnam) in plastic pots (6 × 6 × 4 cm). Each treatment included three replicates of 20 pots (one plant per pot).

Pots were maintained on experimental benches at ambient temperature (18–25 ± 3°C) under a 12-hour photoperiod provided by 120 W Philips lamps, with automatic timing (Panasonic, Japan). Each pot received 5 mL sterile tap water every 2 days and 5 mL Hoagland 1× solution every 7 days. Disease development was assessed by recording the number of diseased plants weekly from 2 to 5 weeks. Symptom onset and progression were also documented.

Disease incidence (DI) and disease severity (DS) were evaluated at 2, 3, 4, and 5 weeks post-inoculation (wpi). DI was calculated as the percentage of symptomatic plants. DS was rated using a 1–4 scale adapted from Prados-Ligero et al. (2007):

1 = no symptoms;

2 = chlorosis of 1–2 basal leaves;

3 = chlorosis/wilt of up to half the plant;

4 = severe wilt or death of the entire plant.

2.6. Data analysis

Morphological traits, including colony pigmentation, mycelial type, conidia size, shape, septation, and other microscopic features, were encoded into a matrix of 41 variables (6 quantitative and 35 binary) (Table S1). Hierarchical cluster analysis was performed using Gower’s distance matrix and Ward’s hierarchical clustering method in R software (R Core Team, 2021) with the function ‘daisy’ from the package ‘cluster’.

For statistical comparisons, Analysis of Variance (ANOVA) was conducted following verification of normality (Shapiro–Wilk test) and homogeneity of variance (Levene's test). Post-hoc Tukey tests were performed at α = 0.05 using R.

3.1. Symptomatology

The disease affects carnations at all growth stages, with newly planted seedlings or cuttings being especially vulnerable. Typical symptoms include basal leaf chlorosis, green wilt, dieback, leaf scorch, stem wilt, and vascular browning. Among these, localized wilting and drying of branches or stems is the most characteristic sign observed in greenhouses. All nine surveyed carnation greenhouses exhibited Fusarium-wilt symptoms, with incidence rates ranging from 4.6–20.3%, resulting in an average incidence of 10.7% (Table 1).

In seedlings, infection begins with yellowing of lower leaves and drooping of younger, pale green leaves. Roots and basal stems are often poorly developed, turning brown to black and drying out. This leads to rapid wilt and dieback. In mature plants, symptoms usually appear on one or a few branches or stems. Unusually, wilt symptoms in carnation do not begin with typical leaf drooping. Instead, they often start with pale discoloration or yellowing and wrinkling at the leaf tip on one side of the stem. This spreads along the leaf and eventually affects the entire plant, causing widespread wilting, scorch, and collapse. The disease often starts at harvest wounds, then spreads downward and into secondary branches/stems, causing whole plants to wither.

As infection progresses, vascular tissues turn partially or completely brown. Stems become dry and blight, with yellow-orange to orange structures resembling Fusarium sporodochia. Stem bases and roots also darken and rot. Chemical treatments applied after symptom onset may slow disease progression but do not restore plant health. Overall, the symptoms align closely with typical Fusarium wilt patterns.

3.2 Morphological Clustering of Fusarium Isolates

A total of 21 Fusarium isolates were obtained from 27 symptomatic carnation plants across nine greenhouses in Lam Dong Province. Based on initial morphological comparison with diagnostic keys from FusaHelp, the Fusarium isolates were preliminarily classified into two species complexes: Fusarium incarnatum-equiseti species complex (FIESC) and F. oxysporum species complex (FOSC) (Table 1). To further evaluate morphological variation among isolates, hierarchical clustering was performed using a mix-type dataset of 41 attributes (35 binary and 6 numerical), encompassing colony pigmentation, mycelial patterns, and conidial and reproductive structures.

Table 1

Disease incidence of *Fusarium* wilt in carnations and the frequency of *Fusarium* spp. recovered from commercial carnation greenhouses in Lam Dong, Vietnam.
Farm	Latitudes	Longitudes	Diseaese Incidence (%)	No. Isolates*	FOSC*	FIESC*	Isolates	Phenotype
1	12°0'8.8"	108°24'31.9"	5.2	2	2	---	C1612 C1611	FOSC I FOSC I
2	11°59'15.4"	108°24'51.1"	10.6	4	1	3	C2132 C2111 C2121 C2131	FOSC I FIESC II FIESC II FIESC II
3	11°57'4.9"	108°24'41"	4.9	1	---	1	C3221	FOSC II
4	11°57'23.6"	108°30'32.6"	12.7	3	---	3	C4911 C4921 C4931	FIESC II FIESC II FIESC II
5	11°57'51.6"	108°30'38.1"	15.9	1	1		C5131	FIESC I
6	11°56'32.6"	108°31'07.0"	20.3	1	1		C5331	FOSC I
7	12°0'30.4"	108°24'55.8"	5.7	3	2	1	C5621 C5631 C5612	FOSC I FOSC I FIESC II
8	12°0'51.1"	108°24'37.4"	4.6	3	3	---	C5811 C5821 C5831	FOSC I FOSC I FOSC I
9	12°0'42"	108°24'21.5"	16.2	3	---	3	C5911 C5912 C5921	FIESC I FIESC I FIESC I
Average			10.7		10	11
Note: FOSC = Fusarium oxysporum species complex; FIESC = F. incarnatum-equiseti species complex; *presented as counts

The resulting dendrogram (Fig. 3A), generated using Gower’s distance matrix and Ward’s hierarchical clustering method, grouped the isolates into two major clusters corresponding to FIESC and FOSC, each further subdivided into two subclusters (FIESC I/II and FOSC I/II). The high agglomerative coefficient (0.99) indicated strong internal consistency in the hierarchical structure, suggesting reliable morphological differentiation among the isolates. Subcluster FOSC I contained the majority of isolates (9 in total), suggesting a relatively high frequency of similar F. oxysporum-like morphotypes within the collection. In contrast, FOSC II comprised only a single isolate (C3221), possibly representing a morphologically distinct or rare variant. FIESC I and II included 4 and 7 isolates, respectively, showing some morphological diversity within the F. incarnatum-equiseti complex as well.

These groupings are summarized in the bar chart (Fig. 3B), showing the distribution of individual isolates across subclusters. This morphology-based clustering supports the preliminary complex-level classification and reveals intra-complex diversity. Detailed comparative descriptions of morphological features across clusters are presented in the following section, while final identification will be confirmed by multilocus phylogenetic analysis using ITS and TEF-1α sequences.

3.3 Morphological characterization

All FIESC isolates grew at 2.8–4.59 (mean = 3.45 ± 0.58) cm/day (in radius) and formed flocculent, circular colonies with irregular edges and abundant aerial hyphae. Colony pigmentation ranged from greenish-yellow or light brown at the center to white or cream toward the margins (Fig. 3a, b, I, j). All did not produce sporodochia until 30 days on CLA (Fig. 3c, k). Conidiophores were both branched (ramified) and unbranched (un-ramified) (Fig. 3d, e, l, m). FIESC I isolates formed oval to fusiform microconidia (0–2 septa), and sparse chlamydospores were observed only in aged cultures (Fig. 3o). In contrast, FIESC II isolates lacked microconidia but produced mesoconidia—smaller, 1–2 septate, macroconidium-like spores (measured as 14.81–27.88 × 2.96–4.55, mean = 21.17 ± 3.94 × 3.73 ± 0.41 µm, n = 40) (Fig. 3f, g). Both groups produced falcate, hyaline macroconidia on mono- and polyphialides, with 3–5 septa, blunt to papillate apical cells, and notched basal cells (Fig. 3f, g, n), distinct from the straighter macroconidia of FOSC. FIESC II macroconidia were broader (23.55–40.4 × 4.31–5.98, mean = 31.55 ± 4.09 × 5.14 ± 0.47 µm, n = 40) than those of FIESC I (23.57–41.28 × 3.8–5.41, mean = 30.91 ± 4.06 × 4.7 ± 0.44 µm, n = 40) (Fig. 3f, g, n), and chlamydospores were absent even after 30 days on CLA, but pseudo-chlamydospores were observed (Fig. 3h).

All FOSC isolates shared key features, including a radial growth rate of 2.57–4.65 (mean = 3.64 ± 0.51) cm/day on PDA and production of microconidia, macroconidia, and chlamydospores (Fig. 3p-af). Sporodochia were bright yellow on CLA (Fig. 3r, z), and microconidia formed in false heads on short, simple monophialides (Fig. 3s, aa). Macroconidia were slightly curved, mostly 3-septate, with parallel sides (Fig. 3u, ac). FOSC I isolates (n = 9) produced off-white, dense colonies with abundant aerial hyphae (Fig. 3x, y). Their microconidia were narrow, elongated 5.05–15.36 × 1.72–3.96 (mean = 8.61 ± 2.43 × 2.45 ± 0.52 µm, n = 40), and maggot-like (Fig. 3ac, ad), while chlamydospores were thick-walled, round to oval (5.1–9.07, mean = 7.04 ± 1.02 µm, n = 40), and either intercalary or terminal (Fig. 3ae, af). In contrast, FOSC II (C3221) had smoother colonies with marginal hyphae growing close to the medium surface (Fig. 3p, q). Microconidia were smaller, mostly aseptate (5.15–11.4 × 2.19–4.38 µm, mean = 7.28 ± 1.54 × 2.95 ± 0.42 µm, n = 40), round to subglobose (Fig. 3u), and chlamydospores (4.49–8.95, mean = 6.56 ± 1.05 µm, n = 40) were mostly terminal with a well-defined curved stalk (Fig. 3v, w).

In summary, these results confirm that the 21 isolates clustered consistently into four morphotypes, including FOSC I, FOSC II, FIESC I, and FIESC II, each with distinctive conidial and colony characteristics.

3.4. Molecular identification

Based on the maximum likelihood (ML) phylogenetic analysis incorporating reference isolates from GenBank, the seven representative isolates from this study were grouped into two distinct clades: the FOSC and the FIESC, clearly separated from other Fusarium lineages (Fig. 4).

Within the FOSC clade, isolates C1611 and C5811 (FOSC I morphotype) clustered together with high bootstrap support (98%) alongside known F. oxysporum strains (e.g., JW11005 and NL 19-94002 from soil in the Netherlands). In contrast, isolate C3221 (FOSC II morphotype) formed a separate subclade with F. nirenbergiae isolates, including NRRL 36261 (banana) and JW289011 (soil, Netherlands), confirming its identity. This subclade was closely related to F. oxysporum, F. odoratissimum, and F. curvatum, supporting F. nirenbergiae as a distinct species within FOSC.

Isolates assigned to FIESC showed clear genetic divergence from FOSC and were distributed into two well-supported subclades. Isolates C5131 and C5912 (FIESC I morphotype) grouped with F. sulawesiense strains from diverse geographic origins and hosts, including cotton (NRRL 34056, USA), rice (LC13720, China), and banana (Indo 186, Indonesia). Meanwhile, isolates C2111 and C5612 (FIESC II morphotype) clustered with F. pernambucanum strains, such as NRRL 32864 (human, USA), NRRL 34070 (cotton, USA), URM 6801 (insect host, Brazil), and LC7019 (banana, China). These results confirm that FIESC I and FIESC II represent two distinct species F. sulawesiense and F. pernambucanum, within the same complex.

In conclusion, the phylogenetic analysis corroborated the morphological identification, distinguishing the 21 isolates into four Fusarium species: F. oxysporum and F. nirenbergiae (FOSC), and F. sulawesiense and F. pernambucanum (FIESC).

3.5 Isolation frequency of Fusarium species

Based on isolation and identification results, four Fusarium species were associated with carnation wilt disease in Lam Dong: F. oxysporum, F. nirenbergiae, F. pernambucanum, and F. sulawesiense, each with varying isolation frequencies. F. oxysporum was the most prevalent, with nine isolates, followed by F. pernambucanum (7 isolates), F. sulawesiense (4), and F. nirenbergiae (1).

Multiple species were sometimes detected at the same site or within the same diseased plant, indicating possible co-infection. For instance, one sample from Farm 2 yielded both F. oxysporum and F. pernambucanum, and both species were also found at Farm 7. However, due to the low frequency of such occurrences, it remains difficult to draw definitive conclusions about their epidemiological significance (Table 1).

3.6 Pathogenicity of Fusarium spp.

Blotter assay

The blotter assay results showed that Fusarium spp. exhibited varying levels of aggressiveness toward carnation during germination and early seedling growth (Figs. 5 and 6).

In the control group, nearly all seeds germinated by 4 dpi. In contrast, germination rates dropped in seeds infected with Fusarium spp., averaging around 70% for the FOSC and 80% for the FIESC. Interestingly, germination improved in FOSC-infected seeds by 8 dpi, while little change was observed in the FIESC group. A significant difference between the two complexes (p < 0.05) indicated distinct effects on germination dynamics (Fig. 6A–B). Despite this divergence, both complexes caused a marked reduction in seed vigor index by 8 dpi (p < 0.05), though no significant difference observed between them (Fig. 6C–D).

At the species level, differences in impact were also observed. At 4 dpi, F. nirenbergiae, F. oxysporum, and F. pernambucanum caused the most severe reductions in germination (about 60–70%), significantly lower than the rate observed in F. sulawesiense-infected seeds (> 80%). While germination in seeds infected with F. oxysporum and F. nirenbergiae recovered slightly by 8 dpi, those infected with F. pernambucanum remained significantly lower (p < 0.05). All four species had similarly detrimental effects on seedling survival (p < 0.05), reducing it by approximately 40% compared to the control (Fig. 6G–H). However, F. pernambucanum caused the most significant reduction in vigor index (p < 0.05), further highlighting species-specific pathogenicity.

Analysis at the isolate level showed that all isolates impacted germination to some extent. At 4 dpi, most isolates reduced germination compared to the control, but only isolate C1611 showed a significant difference (60% vs. 100% in control). By 8 dpi, C1611-infected seeds showed partial recovery (~ 70% germination), whereas seeds infected with isolates C2111 and C4921 remained at significantly lower germination levels. Isolates C5911 and C5921 (both F. sulawesiense) had minimal impact on germination (Fig. 7A–B).

Survival rate differences were more pronounced. Infections with isolates C1611 and C2111 resulted in the lowest survival (~ 35%), significantly lower than the control (100%) (Fig. 7C). Most other isolates also significantly reduced survival, except for C5821, which did not differ from the control (Fig. 7C).

Regarding seed vigor, isolates also showed distinct patterns. Most of the isolates caused a significant reduction in seed vigor index, except for 3 isolates (C1621, C5811 and C5821) of the FOSC and 2 (C5911 and C5921) of the FIESC group. Among them, C1611 (FOSC) and C2111 (FIESC) showed the strongest negative effects in vigor (Fig. 7D). These findings underscore the variability in aggressiveness among isolates, even within the same species complex.

In conclusion, Fusarium spp. significantly reduced carnation seed germination, seedling survival, and seed vigor. However, the severity of these effects varied considerably among species complexes, individual species, and even among isolates, indicating diverse pathogenic behaviors within the Fusarium genus.

Transplant assay

Throughout the experiment, all control plants (water-treated) remained healthy, while most inoculated transplants exhibited varying degrees of disease symptoms. All Fusarium isolates tested were pathogenic, but the onset and progression of symptoms varied by isolate. Wilt symptoms appeared earliest (at 6 days post-inoculation, dpi) in transplants infected with F. oxysporum isolates C1611 (mean = 7.3 ± 1.53 dpi), while symptoms emerged latest (at 25 dpi, mean = 19.7 ± 5.51 dpi) in those infected with isolates C5912. On average, FOSC isolates induced symptoms at 11.1 dpi, about 4 days earlier than those from FIESC isolates (15.6 dpi), indicating a more rapid onset of infection by FOSC.

Disease incidence varied across isolates and time points. By 2 weeks post-inoculation (wpi), FOSC infections showed the highest incidence, led by isolate C1611 (13.3%), while the lowest was seen in C5811-infected plants (1.7%). For FIESC, disease symptoms were observed in only 2.5% of transplants, with the highest incidence recorded for C2111 and C5131 (with 3% each). At 3 wpi, disease incidence increased slightly but rose sharply between 4–5 wpi, revealing clearer differences between isolates. The most rapid increases were observed in C1611 (from 33.1–53.1–91.7%) and C2111 (from 20–33.3–88.3%) over this period. While early-stage incidence (2–4 wpi) significantly differed between these two isolates, their final disease rates were similar (Fig. 8A-D). This suggests a faster onset for C1611 but a more aggressive progression for C2111. Most remaining isolates caused disease at comparable rates, with incidence increasing steadily over time. However, isolates C5811 (F. oxysporum) and C5912 (F. sulawesiense) consistently showed the lowest pathogenicity across all stages. By 5 wpi, disease incidence reached 55% for C3221, while C5612 and C5131 remained significantly lower (~ 40%, p < 0.05). C5811 and C5912 had the lowest final incidence (~ 25%).

Disease severity followed a similar trend, reflecting the non-uniform development of symptoms across treatments. Although symptoms appeared early, severity indices began to diverge only from 3 wpi onward. At this stage, C1611 caused the highest disease severity (1.4), significantly exceeding that of the other isolates (1.0–1.1). C5912 and C5811 exhibited the lowest severity (1.1) and did not differ from the control (p > 0.05). By 5 wpi, disease severity was highest in C1611- and C2111-infected plants, both reaching indices above 3 (of maximum score = 4), with no significant difference between them. Most other isolates caused mild disease (index ~ 1.5), except for C3221, which produced a moderately higher severity index of ~ 2.3. These results highlight distinct patterns of disease development and aggressiveness among isolates.

In conclusion, all tested Fusarium isolates were capable of infecting carnation transplants and inducing wilt symptoms, though the onset, progression, and severity of disease varied among them. This demonstrates variability in isolate pathogenicity toward carnation transplants.

Finally, re-isolation and morphological verification confirmed the identity of each pathogen from both pathogenicity assays, completing Koch’s postulates.

Taken together, all four Fusarium species identified in this study are causal agents of Fusarium wilt in carnations in Lam Dong. Despite differences in virulence among isolates, all are capable of infecting seeds and seedlings, and transplants, reducing germination, causing seedling mortality, and inducing root rot and wilt symptoms similar to those observed under field conditions.

Carnation Fusarium Wilt (CFW), caused by Fusarium spp., is a globally recognized disease (Polat et al., 2022), and has now been identified in Lam Dong province, Vietnam. Despite extensive global documentation, this study represents the first report on the diversity of Fusarium pathogens associated with CFW in Vietnam. Through field surveys in Da Lat and surrounding areas, we observed characteristic symptoms, including leaf yellowing, blight, and stem core discoloration, with an average disease incidence of ~ 10.7% across surveyed greenhouses. These symptoms and incidence rates are consistent with global reports, although currently at a relatively low level. However, the expansion of intensive carnation farming in Lam Dong, a major floriculture hub, may increase the risk of disease spread. Notably, emerging reports of similar symptoms in other crops within the region underscore the broader threat posed by soilborne Fusarium pathogens.

Fusarium identification based solely on morphology remains challenging due to phenotypic plasticity and interspecific similarities (Harish et al., 2023). Thus, this study combined morphological and molecular approaches, including ITS and TEF-1α sequencing, to confirm species identity. Morphology-based hierarchical clustering divided the recovered 21 isolates into two main species complexes, including F. oxysporum species complex (FOSC) and F. incarnatum-equiseti species complex (FIESC), each forming two distinct morphological clades with ~ 17% dissimilarity. Representative isolates were identified as F. oxysporum (9 isolates), F. nirenbergiae (1 isolate), F. pernambucanum (7 isolates), and F. sulawesiense (4 isolates). Although morphological analysis alone cannot precisely resolve species identity, it remains useful for estimating species diversity and reducing reliance on costly molecular diagnostics, particularly in resource-limited settings.

F. oxysporum is a well-documented carnation pathogen (Deng, 2018) and its detection here aligns with global records. In contrast, F. nirenbergiae is a novel finding on carnations. Previous findings have linked it to wilt diseases in hosts such as maple, tomato, passion fruit, and saffron (Aiello et al., 2021; Mirghasempour et al., 2022; S. Zhang et al., 2025; Zhao et al., 2020). This species was herein phylogenetically clustered in a distinct lineage, closely related to F. curvatum and F. oxysporum (Fig. 3). Its unique features, such as terminal chlamydospores with curved stalks and distinct colony morphology, supported its status as a distinct species and confirm this as the first report in Vietnam and the first association with carnations.

F. pernambucanum and F. sulawesiense (members of the FIESC) also displayed distinct morphological features. F. sulawesiense produced chlamydospores and matched previous descriptions from chili, rice, muskmelon, and soybean (Chakrawarti et al., 2025; Liu et al., 2023; Nik Nurnaeimah Nik Muhammad et al., 2024; Wang et al., 2023). In contrast, F. pernambucanum lacked ovoid microconidia and chlamydospores, but produced mesoconidia, consistent with isolates from melon and mango (Hao et al., 2024; Li & Zhang, 2023). Both formed macroconidia resembling those reported in plum leaf blight in China (Lu et al., 2022), with F. pernambucanum showing notably wider forms. Their pathogenic roles have been increasingly recognized since the Fusarium re-classifications by (Lombard et al., 2019) and (Crous et al., 2021), particularly in postharvest rots of tropical fruits such as chili, melon, mango, grape, and banana (Hao et al., 2024; Li & Zhang, 2023; Liu et al., 2023; Suwannarach et al., 2024; Wang et al., 2023, 2024; Zheng et al., 2024), and in insect diseases (da Silva Santos et al., 2019; Diniz et al., 2022; Gonçalves Diniz et al., 2024). F. sulawesiense has also been linked to melon wilt in Malaysia, soybean pod blight in China, and lettuce wilt in Vietnam ((Le et al., 2024; Nik Nurnaeimah Nik Muhammad et al., 2024; Wang et al., 2024). While both species have occasionally co-occurred (e.g., melon rot in Brazil; (Medeiros Araújo et al., 2021), neither had been linked to carnations. This study marks the first record of F. pernambucanum in Vietnam.

In Lam Dong, multiple Fusarium species have been isolated from vegetable and ornamental crops (Le, 2024; Le et al., 2024), with F. oxysporum consistently the most prevalent. FIESC members, including F. compactum and F. sulawesiense, were previously only reported from lettuce (Le et al., 2024). Globally, FIESC has rarely been linked to carnations, with limited reports from New Zealand (Broadhurst, 1990) and Turkey (Atakan & Özkaya, 2020). Thus, our findings expand the known host range of FIESC and highlight the increasing complexity of Fusarium populations affecting carnations in Vietnam.

Previous studies have highlighted regional variation in Fusarium species associated with carnation wilt. In Iran, F. proliferatum and F. solani have been implicated (Fettahi et al., 2014), while in Spain, F. oxysporum, F. proliferatum, and F. solani were reported (Basallote-Ureba et al., 2016). Multiple species have also been identified in Turkey and Iran. In the present study, 21 Fusarium isolates from symptomatic carnations were almost evenly distributed between the FOSC and FIESC complexes (10 and 11 isolates, respectively), suggesting a balanced involvement in disease occurrence. Within each complex, F. oxysporum was predominant in the FOSC group (9 out of 10), while F. pernambucanum represented nearly two-thirds of the FIESC group (33.3% of the total), indicating their likely primary roles in carnation wilt in Vietnam. Co-infections of species were observed at a low rate, complicating efforts to draw significant conclusions about the role of individual species in disease expression. Therefore, further studies using both single and combined inoculation assays are needed to clarify the pathogenic roles of individual species and their interactions in co-infection scenarios.

Pathogenicity assays confirmed that all Fusarium species could infect seeds and transplants, causing varying levels of disease. FOSC isolates generally delayed or suppressed seed germination, while FIESC isolates caused seed necrosis or reduced germination, indicating differing pathogenic profiles between complexes. At later stages, symptoms converged (e.g., seedling browning and root rot, transplant yellowing and wilting), though disease progression and severity varied by isolate. This inter- and intra-species variability complicates detection and management, as pathogenicity does not always correlate with species identity or isolation frequency.

Highly virulent isolates, identified from both species complexes including C1611 (F. oxysporum) and C2111 (F. pernambucanum), maintained their aggressiveness across seed and transplant stages. Interestingly, no significant correlation was observed between isolate virulence at seed and transplant stages (p > 0.05), suggesting organ- or stage-specific pathogenic responses. For example, C3221 was more virulent in transplants than during seed germination, highlighting the importance of evaluating pathogenicity across multiple developmental stages.

These results align with previous studies reporting discordance between frequency and virulence among Fusarium spp. (Atakan & Özkaya, 2020; Fettahi et al., 2014). In Turkey and Iran, F. oxysporum was the most frequently isolated, yet F. solani exhibited greater virulence (Atakan & Özkaya, 2020). Likewise, in Iran, F. solani was more virulent than F. proliferatum (Fettahi et al., 2014). Specific species also exhibit tissue and symptom specificity, for example, F. oxysporum typically affects roots and crowns, while F. proliferatum may be limited to the rhizosphere (Shahbazi et al., 2021b). Also, symptom expression varies geographically and with infecting Fusarium species: in New Zealand, F. graminearum causes dieback, while F. avenaceum induces stem rot (Broadhurst, 1990); in Victoria, F. oxysporum is linked to wilt, F. avenaceum to basal rot, and F. graminearum to stem rot and dieback (Wright et al., 1997). In our observation, carnations exhibited progressive leaf blight, akin to early senescence, resembling symptoms caused by F. oxysporum f. sp. dianthi in Colombia (Poli et al., 2013). Although F. oxysporum isolates in our study may be related to Colombian race 1, as supported by pathogenicity tests, race identification remains challenging due to environmental influences and host’s variable susceptibility (Prados-Ligero et al., 2007).

In Vietnam, carnations are typically grown in plastic-house monocultures for extended periods (> 18 months), promoting the buildup of soilborne pathogens. Infected stock plants can produce asymptomatic but infected cuttings, enabling local transmission before symptom onset (Nelson, 1964). Control is further constrained by the lack of registered fungicides for carnations in Vietnam. Formerly effective chemicals such as methyl bromide and carbendazim (Ben-Yephet et al., 1994) are now banned. Cut flowers can serve as symptomless carriers of Fusarium pathogens (Nelson, 1964). As Vietnamese carnations are exported to over 20 countries, including Australia, the EU, Japan, and Korea, the risk of spreading virulent F. oxysporum or newly emerging FIESC species via symptomless cut flowers presents significant phytosanitary concerns.

In conclusion, this study provides the first comprehensive report on the causal agents of vascular wilt in carnations in Vietnam. Using integrated morphological, molecular, and pathogenicity-based approaches, we identified F. oxysporum, F. nirenbergiae, F. pernambucanum, and F. sulawesiense as responsible pathogens. These findings significantly advance our understanding of Fusarium diversity in carnations and emphasize the need for continued surveillance, improved diagnostics, and phytosanitary measures to safeguard both local production and international trade.

Funding: This work uses the science and technology development fund of Dalat University, Vietnam.

Conflict of interest

The authors declare no conflict of interest.

Data availability statement

Data that support the findings of this study are available from the corresponding author on reasonable request.

Author contributions

D. Le did all this work

Acknowledgements

The authors thank the local carnation farmers for allowing this study to be conducted on their farms. We acknowledge the Ministry of Education and Training of Vietnam for funding a previous project (2023), whose research contributed to this work, and Dalat University for supporting the completion of most of the study.

Ethical Approval Statement

The study does not require local ethics committee approval.

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New Insights into the Diversity and Pathogenicity of Fusarium Species Causing Carnation Wilt in Lam Dong, Vietnam

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

2.1. Survey, sampling and isolation of pathogens

2.2. Morphological Characterization

2.3. Molecular Identification

2.3.1 Genomic DNA extraction and polymerase chain reaction (PCR)

2.3.2 Phylogeny analysis

2.5. Pathogenesis tests

2.5.1 Seed assay

2.5.2 Cutting assays

2.6. Data analysis

RESULTS

3.1. Symptomatology

3.3 Morphological characterization

3.4. Molecular identification

Blotter assay

Transplant assay

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

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