Polyphasic identification of Fusarium species associated with sunflower seed-borne diseases in Brazil

doi:10.21203/rs.3.rs-4159646/v1

Download PDF

Research Article

Polyphasic identification of Fusarium species associated with sunflower seed-borne diseases in Brazil

https://doi.org/10.21203/rs.3.rs-4159646/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Fusarium spp. are among the most common fungal species associated with diseases both on wild and cultivated plants, including sunflowers. They infect all plant tissues causing damage to roots, bundle vessels, stems, leaves, and seeds, often causing significant yield losses. Because contaminated seeds can spread diseases into new areas and transmit them to growing plants, the quality and sanitary status of the seeds are the key to limit the spread of the disease. This study aimed to identify and determine the prevalence of Fusarium species associated with sunflower seeds and access their transmission to growing plants. A set of 49 Fusarium isolates was collected from seeds of eight sunflower cultivars. They were characterized through morphological, cultural, and genetic features. Genetic diversity was estimated through amplification of the elongation factor gene (EF-1 α), which also served to select representative isolates to perform amplification of the β-tubulin 2 gene (TUB2). There were identified four species of Fusarium (i.e., F. fabacearum, F. proliferatum, F. pseudocircinatum and F. verticillioides) that caused seed rot, vascular darkening, withering, malformation, and stunting of growing sunflower plants. Among them, F. proliferatum was the most prevalent species. Our results highlight that various species of Fusarium are associated with damage on sunflower seeds and all of them can be transmitted through infected seeds and cause disease in growing plants.

Helianthus annuus L.

phytopathogenic fungi

plant disease diagnosis

DNA analysis

Sunflower (Helianthus annuus L.) is the fourth main oilseed crop worldwide, along with oil palm (Elaeis guineensis Jacq.), soybean [Glycine max (L.) Merr.], and rapeseed (Brassica napus L.) (Zhang et al., 2018; Pilorgé, 2020; USDA, 2023). Its grains contain about 38–50% of high-quality oil, primarily used for human consumption, as well as feedstock for pharmaceutical, chemical, cosmetic and biofuel industries. They also serve to feed birds, and the sunflower cake that results from oil extraction can integrate diets for feedlot cattle, swine, and poultry (Castro & Leite, 2018). According to U.S Department of Agriculture (USDA), Russia, Ukraine, European Union, Argentina, China and Turkey produced in 2023 over 83% of all the sunflowers in the world (USDA, 2023). Brazil is ranked 18th place in terms of sunflower production, and as indicated in the last Brazilian forecast harvest grain of the 2021/22 crop season, sunflower was cultivated in ca. 39.5 thousand ha and produced ca. 41.1 thousand tons of grains, which represented a ca. 40% increment on both cultivated area and grain production (CONAB, 2022). For 2023, the sunflower area is forecast at 34.7 thousand ha, with a production of ca. 59.4 thousand tons of grains (IBGE, 2023). Nevertheless, several factors must be aligned to increase yield while the cultivated area is reduced, including suitable environmental conditions, the quality and correct dosage of inputs, and the sanitary status of the plants (Naorem et al., 2023).

Like any crop, sunflower can be affected by pathogens (e.g., bacteria, and fungi) that survive in seeds and use them as a pathway of dissemination (Addrah et al., 2020; Chalam et al., 2020). Fungi can reduce seed quality and seed longevity, thus impacting germination and plant establishment in the field (Srinivas et al., 2017). Some of them also produce harmful mycotoxins to plants, humans, and other animals (Martín et al., 2022). Seed-borne diseases associated with Fusarium species, affect numerous cultivated plants, e.g., sunflower, maize, wheat, beans, rice, soybeans, forage grasses, among others (Guo et al., 2016; Ignjatov et al., 2016; Gai et al., 2018; Chang et al., 2020; Costa et al., 2021; Farias et al., 2022; Zhao et al., 2022; Martín et al., 2022; Farias et al., 2023). There were recorded at least 14 species of Fusarium (i.e., F. anthophilum, F. chlamydosporum, F. equiseti, F. incarnatum, F. longipes, F. moniliforme, F. oxysporum, F. pallidoroseum, F. proliferatum, F. pseudocircinatum, F. scirpi, F. solani, F. sporotrichioides, and F. subglutinans) associated with damage on sunflower seeds (Sharfun-Nahar & Mushtaq, 2006; Sharfun-Nahar & Mushtaq, 2007; Keerio et al., 2017; Addrah et al., 2020).

Many Fusarium species are major pathogens in many different crops, however, in sunflowers, Fusarium it was in the past considered a minor pathogen (Tikhonov, 1992; Gulya et al., 1997), while in some studies Fusarium has shown to cause a devastating disease in sunflower (Yakut-Kin, 1995; Antonova et al., 2002). In fact, a very recent study showed that F. pseudocircinatum is the key species causing stunting and malformation in sunflower plants in the north region of Brazil (Farias et al., 2023). Fusarium-associated diseases have emerged as a worrying disease in sunflower in the recent years, causing yield and economic losses for Brazilian growers, and has also been reported in Russia (Gontcharov et al., 2006).

From contaminated seeds, these pathogens can be transmitted to growing plants where they cause root rot, vascular darkening, chlorosis, withering, stunting, and damping off (Keerio et al., 2017; Torres-Cruz et al., 2022). Another major concern related to Fusarium species is their ability to survive in the soil for long period without a host (Keerio et al., 2017).

Traditionally, several fungicides are known to have the potential to control seed-borne fungi such as Fusarium species, however, their use on seeds has been controlled due to their harmful effect on seeds/oil quality as well as on water and related ecosystems (Palti, 1981). In addition, the constant use of fungicides over time results in the prevalence of resistant genotypes (Materatski et al., 2019). Altogether, these problems highlight the need to select and sow healthy seeds, for this, the combination of morphological, cultural and molecular features is important to precisely determine the Fusarium species associated to sunflower, and then, rapidly select seeds free from these pathogenic agents and, consequently, prevent its dissemination (Martín et al., 2022).

Previously, Fusarium identification was performed only through morphological and cultural features (Koyyappurath et al., 2016). However, such plastic characteristics may change due to composition of the culture media, media pH, light presence, and temperature. This led to the adoption of a polyphasic approach that combines morphological and cultural features along with a phylogenetic multilocus analysis of DNA sequences (O’Donnell et al., 2022). In addition, besides the correct identification, it is also important to study the transmission rate of Fusarium seed-borne diseases to growing plants, so the standards and procedures of seed certification can be improved (Kumar et al., 2020). To our best knowledge, this is the first study that applied a polyphasic approach to identify the species of Fusarium associated with sunflower seed-borne diseases. In addition, it was also determined the Fusarium prevalent species and evaluated whether they are transmitted to growing plants.

Sampling and isolation of Fusarium

Fusarium isolates were obtained from sunflower seeds of eight cultivars (Aguará 04, Aguará 06, BRS 321, BRS 322, BRS G26, GF 101, Hélio 250, and Olisun 3) exploited in Alagoinha, Paraíba State, Brazil (06º 57' 00'' S, 35º 32' 42'' W), during the 2017/18 crop season. Seeds were provided by ‘Empresa Brasileira de Pesquisa Agropecuária – EMBRAPA’, Campina Grande, Paraíba State, Brazil. After discarding the damaged ones, a set of 200 seeds were surface disinfected by submersing in a 1.0% aqueous solution of NaClO for 3 min, rinsed three times with sterile water, and left to dry on sterilized filter paper prior subjection to the blotter test (BRASIL, 2009). Seeds were individually transferred to Petri dishes (15.0 cm diameter) double lined with sterilized filter paper moistened with 10 mL of sterile water and incubated for seven days into Biological Oxigen Demand (B.O.D.) chamber set to 25 ± 2°C, 12:12 L:D daylength. By using a sterile histological pin, typical structures of Fusarium that grew over the seeds were transferred to Petri dishes (9.0 cm diameter) previously filled with potato dextrose agar (PDA) media, and incubated for seven days into B.O.D. under similar conditions. Based on morphological features of Fusarium spp. (Leslie & Summerell, 2006), monosporic cultures were established, and the isolates were deposited on a collection at the Phytopathology Laboratory of the ‘Universidade Federal da Paraíba (UFPB)’, Areia, Paraíba State, Brazil.

Morphological characterization

Representative isolates of Fusarium spp. were morphologically characterized following Leslie and Summerell (2006). The pigmentation of 10–14 days old colonies grown at 25 ± 2 ºC on PDA was determined using the mycological color charts of Rayner (1970). In contrast, 10–14 days old colonies grown at 25 ± 2 ºC on synthetic low-nutrient agar (SNA) media added with carnation leaves were used to observe length, width, shape, and septation of micro- and macroconidia (n = 15 of each); disposition of the microconidia (i.e., false head or chains); nature of conidiogenous cells (i.e., mono- and/or polyphialides); and the presence or absence of chlamydospores. Both micro- and macroconidia were measured using the program Motic Images Plus 2.0 in a computer coupled with an Olympus BX53 microscope. Photographs were taken using an Olympus DP73 camera and the program CellSens Dimension at 200, 400 and 1000x magnification. A composite photo plate was assembled from individual photographs to enable the comparison of relevant micromorphological features.

To evaluate the mycelial growth of the representative isolates, a disc (5.0 mm diameter) from each colony was transferred to four Petri dishes (9.0 cm diameter) filled with PDA incubated in the dark at 25 ± 2 ºC. For seven consecutive days, the diameter of the colonies was measured with a digital caliper rule, and the Mycelial Growth Index (MGI) was estimated based in Oliveira (1991) as follows: MGI = (D - Dp)/N, where D is the colony diameter in the current day; Dp is the colony diameter from the previous day; and N is the number of days after inoculation, respectively. The final colony size (FCS) was measured on the 7th day post inoculation.

To estimate the conidia production, a spore suspension was prepared by adding 20 mL of sterile water to 7 days old colonies grown on PDA at 25 ± 2 ºC, which were scraped with a soft paint brush to dislodge the spores. This suspension was filtered through double folded sterile gauze and the spores were counted using a hemocytometer (Alfenas & Mafia, 2016).

All experiments followed a randomized design.

DNA extraction, PCR and DNA sequencing

Genomic DNA extraction from 5–10 days old colonies of each Fusarium isolate were performed using the cetyltrimethylammonium ammonium bromide (CTAB) method (Doyle & Doyle, 1987), with some modifications as described previously (Materatski et al., 2019). The quality and concentration of DNA was determined by using a Quawell Q9000 micro spectrophotometer (Quawell Technology, Beijing, China).

The elongation factor gene (EF-1α) was amplified with Fa + 7 and Ra + 6 primers (Karlsson et al., 2016) to estimate the initial genetic diversity and select representative isolates for amplification of the β-tubulin 2 gene (TUB2). Different haplotypes were identified with DnaSP 4.0 (Rozas et al., 2003), and a haplotype network was generated through PopART v. 1.7 (Leigh & Bryant, 2015) by using the algorithm TCS (Clement et al., 2000; Clement et al., 2002). The representative isolates were chosen considering the sunflower cultivars, and their β-tubulin 2 (TUB2) partial gene region was amplified with T1 and T22 primers (O'Donnel & Cizelnik, 1997).

Polymerase chain reactions (PCR) were performed in a final volume of 25 µL containing a mixture of 7–20 ng of genomic DNA, Tris-HCl 10 mM (pH 8.6), KCl 50 mM, MgCl₂ 1.5 mM, and dNTPs 0.2 mM, 0.2 µM of each primer, and 2.5 U of DreamTaq DNA polymerase. PCR cycles were performed in a thermocycler (BioRad) as follow: initial denaturation at 95 ºC for 2 min, followed by 40 cycles of denaturation at 95 ºC for 30 s, annealing at 67 ºC (EF-1α) or 58 ºC (TUB2) for 50 s, extension at 72 ºC for 60 s and a final extension at 72 ºC for 10 min. The PCR products were separated by electrophoresis in a 1.5% agarose gel with Tris-acetate-EDTA (TAE) 1.0X buffer and were revealed under UV light. These PCR products were purified with DNA Clean & Concentrator (Zymo Research) according to the manufacturer guidelines. DNA sequencing was performed by Macrogen (Madrid, Spain).

Sequence alignment and phylogenetic analysis

Consensus were obtained using the Staden Package (Staden et al., 1998). All obtained consensus sequences were used as queries to the Fusarium-ID database (Torres-Cruz et al., 2022) and NCBI nucleotide database using the blast algorithm (Johnson et al., 2008) to confirm the taxonomic assignments of the isolates. Sequences representing ex-types and related sequences were retrieved from GenBank. Sequences from the present study were deposited in GenBank (Supplementary table S1).

Multiple sequence alignments for each individual locus were estimated online using the G-INS-i strategy in MAFFT version 7 (Katok & Standley, 2013; Katoh et al., 2019), with default parameters for gap opening and extension and 200PAM / κ = 2 nucleotide scoring matrix. Wherever needed, they were manually adjusted in MEGA6 (Tamura et al., 2013).

Phylogenetic analyses were performed using the Maximum Likelihood (ML) and Bayesian Inference (BI) methods for both individual and concatenated genes. ML and BI analyses were performed using RAXML-HCP2 v.7.0.4 (Stamatakis, 2014) and MrBayes v 3.2.1 (Ronquist et al., 2012), respectively, implemented in the CIPRES cluster (https://www.phylo.org/portal2/home.action). ML analyses were carried out with 1000 pseudo replicates (-m GTRGAMMA -p 12345 -k -f a -N 1000 -x 12345) under the GTR-GAMMA model. With respect to BI, evolution models were estimated in MrModeltest 2.3 (Nylander, 2004) using the Akaike information criterion (AIC) for each gene. The combined data set was partitioned to reflect the most appropriate nucleotide substitution model for each of the single gene data sets for the BI of the combined data set. Four Markov Chain Monte Carlo (MCMC) chains were conducted for 107 generations, with samplings every 1000 generations. All parameters convergence were checked through Tracer v 1.5 (Rambaut & Drummond, 2010), and the first 25% generations were discarded as burn-in.

Clades were considered well supported when ML bootstrap support was ≥ 70, and BI posterior probability was ≥ 0.95. Phylogenetic trees were visualized in FigTree v1.4.3 (Rambaut, 2012) and edited in Adobe Illustrator CS6 software (Adobe Systems, USA).

Prevalence of Fusarium species and Seed-plant transmission disease bioassay

The prevalence of Fusarium species obtained was determined by calculating the Isolation Rate (IR) as proposed by Veloso et al. (2018): IR (%) = (Cx / Ct) x 100, where Cx is the number of isolates belonging to one species and Ct is the number of isolates per cultivar.

A randomized design bioassay was carried out to access the transmission of Fusarium-associated diseases from sunflower seeds to growing plants. Sunflower seeds, cv BRS 324, from the 2018/19 crop season, were previously tested to measure seed germination and sanity status according to Brasil (2009). They exhibited 93% germination with no incidence of Fusarium species, thus being eligible for the transmission bioassay performed following the methodology described by Sousa et al. (2008). Briefly, 100 surface disinfected (as above) sunflower seeds were distributed over 7 days old colonies of Fusarium representative isolates grown at 25 ± 2°C and 12:12 L:D daylength on PDA amended with mannitol to a -1.0 MPa osmotic potential. Another set of 100 disinfected seeds distributed over PDA + mannitol without fungi served as the control treatment. After exposure for 48 h, the seeds were sown 2.0 cm deep into plastic vases (5.0 L of volume) filled with the sterile commercial substrate Brasplant® and vermiculite (2:1 v/v). They were watered twice a day for 35 days inside a greenhouse with night temperature about 16–18 ºC and day temperature about 28–32 ºC.

Symptomatic growing plants were tallied throughout the experiment, whereas ungerminated seeds were surface disinfected (as described above) and kept into a humid chamber for seven days to observe whether Fusarium species were present. By the 35th day after sowing, all plants were harvested and roots were cleaned with tap water. From each plant, there were taken three fragments of roots, stems, and leaves, which were surface disinfected by submersing in 70% ethanol for 30 s, 1.5% NaClO aqueous solution for 1 min, rinsed three times with sterile water, and dried on sterilized filter paper. These fragments were distributed on PDA incubated at 25 ± 2°C and 12:12 L:D daylength for seven days, and the transmission of Fusarium-associated seed-borne diseases was confirmed whenever typical structures of Fusarium grew over them.

The incidence of Fusarium-associated seed-borne diseases on sunflower was calculated as follows: Disease Incidence (%) = (n / N) × 100, where n is the number of symptomatic plants + the number of ungerminated seeds contaminated with Fusarium spp., and N is the total number of examined plants and seeds. The transmission rate (TR) of Fusarium spp. from sunflower seeds to growing plants was calculated after adapting an equation proposed by Teixeira and Machado (2003): TR (%) = (IR / IS) x 100, where IR is the infection rate of Fusarium spp. on sunflower seeds, roots, stems, and/or leaves fragments and IS is the incidence of each isolate on artificially inoculated seeds.

Statistical analysis

Data from mycelial growth, conidia production, and transmission bioassays were checked for normality and homoscedasticity of variance using the Shapiro-Wilk’s and Levene’s tests, respectively. Dependent variables that passed these tests were analyzed by one-way ANOVA and means were grouped by Scott-Knott test (p < 0.05) using the R software statistical package (R Core Team, 2020).

Sampling and isolation

A total of 49 Fusarium isolates was collected from seeds of eight sunflower cultivars. Most of them were collected from cv. Olisun 3 and cv. Hélio (9 each), followed by cv. Aguará 04 (8), cv. BRS 322 (7), cv. BRS 321 (6), cv. GF 101 (5), cv. Aguará 06 (4), and cv. BRS G26 (2) (Supplementary table S2).

Morphological characterization

Regardless of the Fusarium species, all isolates grew aerial mycelium with varying pigmentation, and exhibited different micromorphological features (Fig. 1–2; Supplementary table S3). The ones identified as F. pseudocircinatum grew orange colonies (F4; Fig. 1), and produced straight to slightly curved, hyaline, 3-5-septate macroconidia (31.8–57.0 x 3.7–4.5 µm) tapering towards both ends (Fig. 2A). They produced abundant non-septate, oval shaped microconidia (6.3–8.3 x 2.7–3.4 µm) on false heads; formed mono- and/or polyphialidic short conidiogenous cells (Fig. 2G); and rarely exhibited coiled sterile hyphae (Fig. 2F). There was no chlamydospore (Supplementary table S3).

The isolates retrieved as F. fabacearum produced violet colonies (F5 and F6; Fig. 1), and slightly curved, hyaline, 3-5-septate macroconidia (22.1–41.1 x 3.5–4.2 µm) tapering towards both ends (Fig. 2B). They also produced ellipsoidal, non-septate microconidia (5.9–9.1 x 1.4–2.2 µm) on false heads; conidiogenous monophialidic cells reduced to short single phialide (Fig. 2I); and single, terminal rough-walled chlamydospores in chains (Fig. 2J-L) (Supplementary table S3).

Fusarium verticillioides (isolates formed violet, lilac, light pink, orange, and light orange colonies (Fig. 1). They exhibited slender and slightly falcate 3-5-septate (24.1–49.6 x 2.5-4 µm), or rarely 5-8-septate (35.4–71.4 x 1.5–4.2 µm) macroconidia, respectively (Fig. 2C-D); oval flat-base shaped, non-septate microconidia (6.0-11.9 x 1.5–3.1 µm) on false heads and/or chains; and conidiogenous monophialidic long cells in pairs (Fig. 2H). There was no chlamydospore (Supplementary table S3).

Isolates nested with F. proliferatum grew plentiful white mycelium that turned lilac, light pink, yellow, light yellow or beige (Fig. 1). They produced abundant slightly straight, 3-5-septate macroconidia (23.4–46.5 x 1.9–5.1 µm) (Fig. 2E); oval, non-septate microconidia (5.3–10.4 x 1.5–4.1 µm) on false heads or short chains; and conidiogenous mono- and/or polyphialidic cells (Fig. 2G). Like the prior species, no chlamydospore was observed (Supplementary table S3).

The MGI (1.6–2.6 cm.day − 1) varied significantly among the representative isolates of Fusarium spp. The highest MGI value was obtained with the isolate F6 (F. fabacearum), the lowest with F38 (F. proliferatum) (p = 0.0043), and intermediate values with the others. Likewise, there were significant differences among the isolates with respect to the FCS (4.77-8.00 cm diameter) and the numbers of spores produced (1.4–28.1 x 106 conidia.ml 1) (Fig. 3).

Phylogenetic analysis

There were seven EF-1α haplotypes (H1-H7) among the 49 isolates based on similarity to representative sequences of Fusarium species in the NCBI database. According to BLAST, 43 isolates (H1-H6) were assigned to the Fusarium fujikuroi Species Complex (FFSC), and the other 6 (H7) to the Fusarium oxysporum Species Complex (FOSC) (Fig. 4; Supplementary table S2).

The Fusarium representative isolates (n = 20) obtained from sunflower seeds were distributed among two clades strongly supported by both ML and BI analysis in the concatenated tree, and another clade with strong support only on BI. From the 18 isolates belonging to FFSC, one formed a clade with F. pseudocircinatum, eight grouped with F. proliferatum, and nine were retrieved as F. verticillioides. On the other hand, the two isolates from FOSC were identified as F. fabacearum with significant support in the BI analysis (Figs. 5 and 6).

Prevalence of Fusarium species and Seed-plant transmission disease bioassay

Overall, F. proliferatum was the most prevalent species among 49 Fusarium isolates collected from seeds of eight sunflower cultivars. It represented about 48% of the isolates, whereas F. verticillioides represented 32%, F. fabacearum 12%, and F. pseudocircinatum 4%, respectively (Fig. 7).

All identified Fusarium species were transmitted from seeds to growing plants and caused symptoms that included seed rot (Fig. 8A), malformation (Fig. 8B), vascular darkening (Fig. 8C), withering (Fig. 8D), and stunting. No symptoms were observed on control plants. Due to the progress of the disease, symptomatic seedlings did not survive.

The SG ranged from 80.0 ± 0.70 in the isolate F12 (F. verticillioides) to 64.0 ± 0.40 in the isolate F29 (F. proliferatum). ANOVA analysis revealed low variability of sunflower seeds germination between all Fusarium isolates with no significant differences (p > 0.05); Table 1) in the SG.

Table 1

Per cent germination of sunflower seeds (SG), cv. BRS 324, inoculated or not (= control) with different *Fusarium* species isolates, incidence of the disease on growing plants (ID), and transmission rate (TR) of the disease from seeds to growing plants.
Fusarium species	Isolate	SG (%)	ID (%)	TR (%)
Control	-	80.0 ± 0.70 a	0.0 ± 0.00 d	0.0 ± 0.00 d
F. pseudocircinatum	F4	70.0 ± 0.86 a	13.5 ± 0.64 c	14.0 ± 0.77 c
F. fabacearum	F5	66.0 ± 1.32 a	26.0 ± 0.86 b	27.3 ± 1.27 b
F. fabacearum	F6	70.0 ± 1.32 a	32.0 ± 1.08 b	35.5 ± 1.02 b
F. verticillioides	F11	73.0 ± 0.85 a	39.5 ± 1.88 a	43.8 ± 1.74 a
F. verticillioides	F12	80.0 ± 0.70 a	18.0 ± 0.95 c	19.1 ± 0.39 c
F. verticillioides	F13	71.0 ± 2.01 a	50.0 ± 2.10 a	52.6 ± 2.22 a
F. verticillioides	F18	75.0 ± 0.62 a	26.0 ± 1.25 b	26.0 ± 0.79 b
F. verticillioides	F20	64.0 ± 0.57 a	21.0 ± 1.03 b	21.0 ± 0.42 b
F. verticillioides	F21	78.0 ± 1.19 a	15.0 ± 1.49 c	16.7 ± 1.60 c
F. verticillioides	F22	68.0 ± 0.81 a	13.0 ± 1.10 c	13.0 ± 0.85 c
F. verticillioides	F23	65.0 ± 0.75 a	24.0 ± 0.70 b	24.7 ± 1.22 b
F. verticillioides	F24	77.0 ± 0.75 a	31.0 ± 1.49 b	32.6 ± 1.27 b
F. proliferatum	F28	68.0 ± 1.08 a	14.0 ± 1.55 c	14.6 ± 1.12 c
F. proliferatum	F29	64.0 ± 0.40 a	21.0 ± 0.85 b	21.2 ± 0.96 b
F. proliferatum	F30	67.0 ± 0.62 a	28.0 ± 0.91 b	29.7 ± 1.13 b
F. proliferatum	F36	70.0 ± 0.70 a	31.0 ± 0.28 b	31.3 ± 0.50 b
F. proliferatum	F38	67.0 ± 0.64 a	24.0 ± 1.32 b	24.0 ± 1.14 b
F. proliferatum	F39	72.0 ± 0.70 a	6.0 ± 0.28 c	7.5 ± 0.50 c
F. proliferatum	F46	74.0 ± 0.64 a	38.0 ± 1.32 a	41.5 ± 1.14 a
F. proliferatum	F48	68.0 ± 0.70 a	25.0 ± 0.47 b	25.7 ± 0.85 b
V.C. (%)	-	11.35	28.01	28.14
Per cent values followed by the same letter within columns are similar by Scott-Knott’s test (p < 0.05).

The ID ranged from 50.0 ± 2.10 in the isolate F13 (F. verticillioides) to 6.0 ± 0.28 in the isolate F39 (F. proliferatum). ANOVA analysis showed significant higher ID values to the isolates F13 (F. verticillioides), F11 (F. verticillioides) and F46 (F. proliferatum) (p < 0.05); (Table 1) compared to all others isolates.

Similar results were found in TR, that ranged from 52.6 ± 2.22 isolate F13 (F. verticillioides) to 7.5 ± 0.50 isolate F39 (F. proliferatum). ANOVA analysis also showed significant higher TR values to the isolates F13, F11 and F46 (p < 0.05); (Table 1) compared to all others isolates.

The present study showed, for the first time, the adoption of a polyphasic approach that combines morphological, cultural features, multilocus phylogenetic analyses along with transmission rate of several Fusarium isolates in eigth sunflower cultivars from seed-borne to growing plants. These results highlighted four Fusarium species as a key species associated with seed-borne diseases on sunflower crop in an important producing region in Brazil. Three of them, namely F. pseudocircinatum, F. verticillioides and F. proliferatum exhibited morphological features alike species from the FFSC, whereas F. fabacearum matched those that belong to the FOSC (Leslei & Summerell, 2006). The FFSC species are distributed worldwide and infect a wide range of plants. For instance, F. pseudocircinatum was recently reported causing stunting and malformation in sunflower plants in Brazil (Farias et al., 2023), although it is also indicated as the causal agent of stem-rot vanilla (Vanilla planifolia) in Indonesia (Pinaria et al., 2010), death of acacia (Acacia koa) in Hawaii (Shiraishi, et al., 2012), malformation of mango trees (Mangifera indica) in Mexico, Dominican Republic, and Australia (Freeman et al., 2014; García-López et al., 2016; Liew et al., 2016), besides lateral sprouting and internode shortening of mahogany (Swietenia macrophylla) in Mexico (Santillán-Mendoza et al., 2018). Despite the F. pseudocircinatum showed lower prevalence in the eight cultivar (4%) and the isolate (F4) presenting lower percentage values of ID and TR, the present results seem to suggest that sunflower seeds of the cultivars; Aguará 06, BRS 321, BRS 322, BRS G26, GF 101 and Hélio 250 may be able to present some resistance characteristics to this Fusarium species, since none of these cultivars seeds showed a positive infection and transmission of this isolate, however, specific studies must be carried out to confirm these results. In agreement with the F. pseudocircinatum results, five cultivars; Aguará 06, BRS 321, BRS 322, BRS G26, and GF 101 seem to also present some resistance to the FOSC species in our study, F. fabacearum (isolates F4 and F5). The F. fabacearum species is often associated with vascular bundle darkening, root rot, wilt, and defoliation followed by death of Malay apple trees (Sygium malaccensis), a common plant species in the north and northeast of Brazil (Farias et. al., 2021). Although, our results suggest that both F. pseudocircinatum and F. fabacearum species appear to have a lower impact in sunflower seeds.

In contrast, F. proliferatum and F. verticillioides were the most prevalent species from the different cultivars used in the present study (48% and 32%, respectively), in addition, isolates from both species showed significant higher values of ID and TR, placing F. proliferatum and F. verticillioides as the key species causing stunting and malformation in sunflower in Brazil. Previous studies had already demonstrated that F. proliferatum causes sunflower withering (Sharfun-Nahar & Mushtaq, 2006), but also deterioration of cowpea seeds (Vigna unguiculata) (Ignjatov et al., 2016) and systemic colonization of wheat plants and contaminated the grains with fumosinins and beauvericin (Guo et a., 2016). Furthermore, it is already well documented that F. verticillioides causes significant damage to rice and maize seeds, which impacts germination, plant emergence (Bashyal et al., 2016; Fantazzini et al., 2016; Souza et al., 2022), and grain storage (Gai et al., 2018). This species it is also associated with pre- and postemergence damping off, root rot, and hypocotyl necrosis of ‘guajuvira’ (Cordia americana), a forest species widely distributed in Brazil (Walker et. al., 2016). Both, F. verticillioides and F. proliferatum are often asymptomatic seed-borne pathogens of maize and produce harmful mycotoxins to humans and other animals (Gaige et al., 2020). As seen previously, the identified species of Fusarium may infect and cause damage to several crops. Based on that and considering that sunflower is commonly cultivated in combination with or in succession to maize, beans, and soybeans in Brazil, the adoption of different preventive measures are crucial to avoid cross-infections among Fusarium species on these plants. For example, the use of sunflower tolerant/resistant varieties to Fusarium species, crop rotation, in combination with or in succession, with species of low susceptibility to Fusarium species, combined with the use of certified and healthy sunflower seeds are determining to avoid cross-infections among Fusarium species on these plants.

Besides causing rot of sunflower seeds, the present results showed that all identified species were transmitted, in some cultivars the case of F. pseudocircinatum and F. fabacearum or in all cultivars the case of F. proliferatum and F. verticillioides, to growing plants and inflicted diseases with symptoms that included malformation, vascular darkening, withering, and stunting and as the disease progressed, the symptomatic plants died. These results corroborate Machado et al. (2013), who argue that high incidence of Fusarium spp. on seeds, under favorable conditions, may affect seed germination and plant development in the field. It must be stressed that seeds are among the most important agricultural inputs, and its genetic and sanitary qualities are the keys to determine the success or failure of the crop. In addition, the high incidence of the disease on growing plants (ID), and transmission rate (TR) of the Fusarium species showed in the present results may be associated with several factors. The Fusarium species are known to survive for long periods in the soil (Keerio et al., 2017), this characteristic combined with the ability to infect several hosts, and the appearance of resistant genotypes due to the indiscriminate use of fungicides may be the reason for the high ID and TR values. Currently, there is no established sanitary standard in Brazil for certification of sunflower seeds, and considering the pathogenicity and high transmissibility of Fusarium, a single contaminated seed is enough to condemn a whole seed lot. To establish a solution measure for this gap, the present study revealed that a polyphasic approach is suitable to identify the species of Fusarium species associated with sunflower seeds, which may guide protocols to improve plant disease management programs. We also showed that F. proliferatum was the most prevalent species in our collection, and future works based in the development of control measures should target this species.

Acknowledgment

Patrick Materatski is supported by Portuguese National Funds through FCT/MCTES under the CEEC contract (2021.01553.CEECIND/CP1670/CT0003). Mônica Danielly de Mello Oliveira is supported by CAPES PDPG-FAPIII under the contract 88887.929825/2023-00.

Conflict of interest statement

The authors declare no conflict of interest.

Addrah, M. E., Zhang, Y., Zhang, J., Liu, L., Zhou, H., Chen, W., & Zhao, J. (2019). Fungicide treatments to control seed-borne fungi of sunflower seeds. Pathogens, 9(1), 29. https://doi.org/10.3390/pathogens9010029
Alfenas, A. C., Mafia, R. G. (2016). Métodos em Fitopatologia. UFV Books.
Bashyal, B. M., Aggarwal, R., Sharma, S., Gupta, S., Rawat, K., Singh, D., Singh, A. K., & Krishnan, S. G. (2016). Occurrence, identification and pathogenicity of Fusarium species associated with bakanae disease of basmati rice in India. European Journal of Plant Pathology, 144(2), 457-466. https://doi.org/10.1007/s10658-015-0783-8
Bell, A. A., Gu, A., Olvey, J., Wagner, T. A., Tashpulatov, J. J., Prom, S., Quintana, J., Nichols, R. L., & Liu, J. (2019). Detection and characterization of Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) isolates of diverse geographic origins. Plant Disease, 103(8), 1998-2009. https://doi.org/10.1094/PDIS-09-18-1624-RE
Brasil. Ministério da Agricultura Pecuária e Abastecimento. (2009). Manual de análise sanitária de sementes. MAPA/ACS Books.
Catão, H. C. R. M., Aquino, C. F., Sales, N. D. L. P., Soares, E. P. S., Caixeta, F., & Martinez, R. A. S. (2016). Sunflower seeds quality stored in uncontroled conditions, treated with hydrolats and vegetable extracts. International Journal of Current Research, 8(9), 39295-39300.
Chalam, V. C., Deepika, D. D., Abhishek, G. J., & Maurya, A. K. (2020). Major seed-borne diseases of agricultural crops: International Trade of Agricultural Products and Role of Quarantine. In R. Kumar, & A. Gupta (Eds.), Seed-Borne Diseases of Agricultural Crops: Detection, Diagnosis & Management (1st, pp. 25-61). Springer.
Chang, X., Li, H., Naeem, M., Wu, X., Yong, T., Song, C., Liu, T., Chen, W., & Yang, W. (2020). Diversity of the seedborne fungi and pathogenicity of Fusarium species associated with intercropped soybean. Pathogens, 9(7), 531. https://doi.org/10.3390/pathogens9070531
Clement, M., Posada, D. C. K. A., & Crandall, K. A. (2000). TCS: a computer program to estimate gene genealogies. Molecular Ecology, 9(10), 1657-1659. https://doi.org/10.1046/j.1365-294x.2000.01020.x
Conab - Companhia Nacional de Abastecimento. (2022). Acompanhamento da Safra Brasileira de Grãos (v. 9, safra 2021/22, n. 11 décimo primeiro levantamento, agosto 2022). Brasília.
Costa, M. M., Melo, M. P., Carmo, F. S., Moreira, G. M., Guimarães, E. A., Rocha, F. S., Costa, S. S., Abreu, L. M., & Pfenning, L. H. (2021). Fusarium species from tropical grasses in Brazil and description of two new taxa. Mycological Progress, 20(1), 61-72. https://doi.org/10.1007/s11557-020-01658-5
Doyle, J.; Doyle, J. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem., 19(1), 11–15.
Elmer, W. H., Summerell, B. A., Burgess, L. W., & Nigh Jr, E. L. (1999). Vegetative compatibility groups in Fusarium proliferatum from Asparagus in Australia. Mycologia, 91(4), 650-654. https://doi.org/10.2307/3761251
Fantazzini, T. B., Guimarães, R. M., Clemente, A. C. S., Carvalho, E. R., & Machado, J. C. (2016). Fusarium verticillioides inoculum potential and its relation with the physiological stored corn seeds quality. Bioscience Journal, 32(5), 1254-1262.
Farias, O. R., Cruz, J. M. F. D. L., Silva Veloso, J., Souza, J. T., Duarte, I. G., Barbosa, P. R. R., Varanda, C. M. R., Materatski, P., Félix, M. R. F, Oliveira, M. D. M., & Nascimento, L. C. (2023). Fusarium pseudocircinatum causing stunting and malformation of sunflower plants in Brazil. Plant Disease, 107(1), 216. https://doi.org/10.1094/PDIS-01-22-0212-PDN
Farias, O. R., Cruz, J. M. F. L., Veloso, J. S., Duarte, I. G., Barbosa, P. R. R., Félix, M. R. F., Varanda, C. M. R., Materatski, P., Oliveira, M. D. M., & Nascimento, L. C. (2022). Occurrence of Fusarium proliferatum causing vascular wilt on cowpea (Vigna unguiculata) in Brazil. Plant Disease, 106(7), 1992. https://doi.org/10.1094/PDIS-04-21-0839-PDN
Freeman, S., Otero-Colina, G., Rodríguez-Alvarado, G., Fernández-Pavía, S., Maymon, M., Ploetz, R. C., Aoki, T., & O'Donnell, K. (2014). First report of mango malformation disease caused by Fusarium pseudocircinatum in Mexico. Plant Disease, 98(11), 1583. https://doi.org/10.1094/PDIS-04-14-0375-PDN
Gai, X., Dong, H., Wang, S., Liu, B., Zhang, Z., Li, X., & Gao, Z. (2018). Infection cycle of maize stalk rot and ear rot caused by Fusarium verticillioides. PloS One, 13(7), e0201588. https://doi.org/10.1371/journal.pone.0201588
García-López, E., Mora-Aguilera, J. A., Nava-Diaz, C., Villegas-Monter, A., Tovar-Pedraza, J. M., Serra, C., & Batista-Marte, C. M. (2016). First report of Fusarium pseudocircinatum causing mango malformation disease in Dominican Republic. Plant Disease, 100(7), 1501-1501. https://doi.org/10.1094/PDIS-12-15-1397-PDN
Gaur, A., Kumar, A., Kiran, R., & Kumari, P. (2020). Importance of seed-borne diseases of agricultural crops: Economic losses and impact on society. In R. Kumar, & A. Gupta (Eds.), Seed-Borne Diseases of Agricultural Crops: Detection, Diagnosis & Management (1st, pp. 3-23). Springer.
Guo, Z., Pfohl, K., Karlovsky, P., Dehne, H. W., & Altincicek, B. (2016). Fumonisin B1 and beauvericin accumulation in wheat kernels after seed-borne infection with Fusarium proliferatum. Agricultural and Food Science, 25(2), 138-145. https://doi.org/10.23986/afsci.55539
Ignjatov, M., Popović, T., Milošević, D., Vasić, M., Nikolić, Z., Tamindžić, G., & Ivanović, Ž. (2016). Occurrence, identification and phylogenetic analysis of Fusarium proliferatum on bean seed (Phaseolus vulgaris L.) in Serbia. Ratar. Povrt., 53(2), 42-45. https://doi.org/10.5937/ratpov53-9984
Johnson, M., Zaretskaya, I., Raytselis, Y., Merezhuk, Y., McGinnis, S., & Madden, T. L. (2008). NCBI BLAST: a better web interface. Nucleic Acids Research, 36(Web server issue), W5-W9. https://doi.org/10.1093/nar/gkn201
Karlsson, I., Edel-Hermann, V., Gautheron, N., Durling, M. B., Kolseth, A. K., Steinberg, C., Persson, P., & Friberg, H. (2016). Genus-specific primers for study of Fusarium communities in field samples. Applied and Environmental Microbiology, 82(2), 491-501. https://doi.org/10.1128/AEM.02748-15
Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution, 30(4), 772-780. https://doi.org/10.1093/molbev/mst010
Katoh, K., Rozewicki, J., & Yamada, K. D. (2019). MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics, 20(4), 1160-1166. https://doi.org/10.1093/bib/bbx108
Keerio, A., Nizamani, A. Z., Hussain, S., Rafiq, M., Iqbal, S., & Keerio, A. U. D. (2017). Efficacy of some chemical fungicides against fusarium wilt of sunflower in-vitro condition course by Fusarium oxysporum. International Journal of Botany Studies, 2(5), 80-85.
Koyyappurath, S., Atuahiva, T., Le Guen, R., Batina, H., Le Squin, S., Gautheron, N., Edel Hermann, V., Peribe, J., Jahiel, M., Steinberg, C., Liew, E. C. Y., Alabouvette, C., Besse, P., Dron, M., Sache, I., Laval, V., & Grisoni, M. (2016). Fusarium oxysporum f. sp. radicis‐vanillae is the causal agent of root and stem rot of vanilla. Plant Pathology, 65(4), 612-625. https://doi.org/10.1111/ppa.12445
Kumar, A., Gaur, A., Gurjar, M. S., Kumari, P., & Kiran, R. (2020). Seed Health Testing and Seed Certification. In R. Kumar, & A. Gupta (Eds.), Seed-Borne Diseases of Agricultural Crops: Detection, Diagnosis & Management (1st ed., pp. 795-808). Springer.
Leigh, J. W., & Bryant, D. (2015). POPART: full-feature software for haplotype network construction. Methods in Ecology and Evolution, 6(9), 1110-1116. https://doi.org/10.1111/2041-210X.12410
Leslie, J. F., & Summerell, B. A. (2006). The Fusarium laboratory manual. Blackwell Publishing.
Liew, E. C. Y., Laurence, M. H., Pearce, C. A., Shivas, R. G., Johnson, G. I., Tan, Y. P., & Summerell, B. A. (2016). Review of Fusarium species isolated in association with mango malformation in Australia. Australasian Plant Pathology, 45(6), 547-559.
Lombard, L., Sandoval-Denis, M., Lamprecht, S. C., & Crous, P. W. (2019). Epitypification of Fusarium oxysporum – clearing the taxonomic chaos. Persoonia, 43(1), 1-47. https://doi.org/10.3767/persoonia.2019.43.01
Lustri, E. A., Silva, B. T., Peruchi, D. R. E., Moura, I. A., & Fluminhan, A. (2017). Evaluation of agronomic performance of sunflower cultivars (Helianthus annuus L.) during the off-season cultivation at the Western São Paulo region. Periódico Eletrônico Fórum Ambiental da Alta Paulista, 13(1), 37-51.
Machado, J. D. C., Machado, A. Q., Pozza, E. A., Machado, C. F., & Zancan, W. L. A. (2013). Inoculum potential of Fusarium verticillioides and performance of maize seeds. Tropical Plant Pathology, 38(3), 213-217. https://doi.org/10.1590/S1982-56762013000300005
Materatski, P., Varanda, C., Carvalho, T., Dias, A. B., Campos, M. D., Gomes, L., Nobre, T., Rei, F., & Félix, M. R. (2019). Effect of long-term fungicide applications on virulence and diversity of Colletotrichum spp. associated to olive anthracnose. Plants, 8(9), 311. https://doi.org/10.3390/plants8090311
Mukhtar, I. (2009). Sunflower disease and insect pests in Pakistan: A review. African Crop Science Journal, 17(2), 109-118. https://doi.org/ 10.4314/acsj.v17i2.54204
Naorem, A., Jayaraman, S., Dang, Y. P., Dalal, R. C., Sinha, N. K., Rao, C. S., & Patra, A. K. (2023). Soil constraints in an arid environment—challenges, prospects, and implications. Agronomy, 13(1), 220. https://doi.org/10.3390/agronomy13010220
Nylander, J. A (2004). A. MrModeltest v2 (Program distributed by the author). Uppsala University Books.
O’Donnell, K., Whitaker, B. K., Laraba, I., Proctor, R. H., Brown, D. W., Broders, K., Kim, H., McCormick, S. P., Busman, M., Aoki, T., Torres-Cruz, T. J., & Geiser, D. M. (2022). DNA sequence-based identification of Fusarium: A work in progress. Plant Disease, 106(6), 1597-1609. https://doi.org/10.1094/PDIS-09-21-2035
O'Donnell, K., & Cigelnik, E. (1997). Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution, 7(1), 103-116. https://doi.org/10.1006/mpev.1996.0376
Oliveira, J. A. (1991). Efeito do tratamento fungicida em sementes no controle de tombamento de plântulas de pepino (Cucumis sativus L.) e pimentão (Capsicum annum L.).111 p. Dissertação (Mestrado em Fitossanidade) Universidade Federal de Lavras, Lavras, MG.
Palti J. (1981). Cultural Practices, and Infectious Crop Disease. Springer Varlag Books.
Pinaria, A. G., Liew, E. C. Y., & Burgess, L. W. (2010). Fusarium species associated with vanilla stem rot in Indonesia. Australasian Plant Pathology, 39(2), 176-183. https://doi.org/10.1071/ AP09079
Rambaut, A. (2012). FigTree v1.4.0: Tree Figure Drawing Tool. Retrieved March 15, 2022, from https://tree.bio.ed.ac.uk/software/figtree
Rambaut, A., & Drummond, A. (2010). Tracer v. 1.4. Retrieved October 10, 2023, from https://beast.bio.ed.ac.uk/Tracer
Ramos, D. P., Barbosa, R. M., Vieira, B. G. T. L., Panizzi, R. D. C., & Vieira, R. D. (2014). Fusarium graminearum and Fusarium verticillioides infection on maize seeds. Pesqui. Agropec. Trop., 44(1), 24-31. https://doi.org/10.1590/S1983-40632014000100011
Rayner, R. W. (1970). A Mycological Colour Chart. British Mycological Society and CAB International Mycological Institute.
Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M. A., & Huelsenbeck, J. P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61(3), 539-542. https://doi.org/10.1093/sysbio/sys029
Rozas, J., Sánchez-Delbarrio, J. C., Messeguer, X., & Rozas, R. (2003). DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics, 19(18), 2496-2497. https://doi.org/10.1093/bioinformatics/btg359
Santillán-Mendoza, R., Fernández-Pavía, S. P., O’Donnell, K., Ploetz, R. C., Ortega-Arreola, R., Vázquez-Marrufo, G., & Rodríguez-Alvarado, G. (2018). A novel disease of big-leaf mahogany caused by two Fusarium species in Mexico. Plant Disease, 102(10), 1965-1972. https://doi.org/10.1094/PDIS-01-18-0060-RE
Shade, A., Jacques, M. A., & Barret, M. (2017). Ecological patterns of seed microbiome diversity, transmission, and assembly. Current Opinion in Microbiology, 37, 15-22. https://doi.org/10.1016/j.mib.2017.03.010
Sharfun-Nahar, & Mushtaq, M. (2007). Pathogenic effects and transmission studies of seed-borne Fusarium species in sunflower. Pakistan Journal of Botany, 39(2), 645-649.
Sharfun-Nahar, & Mushtaq, M. (2006). Pathogenecity and transmission studies of seed-borne Fusarium species (sec. Liseola and Sporotrichiella) in sunflower. Pakistan Journal of Botany, 38(2), 487-492.
Shiraishi, A., Leslie, J. F., Zhong, S., & Uchida, J. Y. (2012). AFLP, pathogenicity, and VCG analyses of Fusarium oxysporum and Fusarium pseudocircinatum from Acacia koa. Plant Disease, 96(8), 1111-1117. https://doi.org/10.1094/PDIS-06-11-0491
Sousa, M. V., Machado, J. C., Pfenning, L. H., Kawasaki, V. H., Araújo, D. V., Silva, A. A., & Martini Neto, A. (2008). Methods of inoculation and effects of Fusarium oxysporum f. sp. vasinfectum in cotton seeds. Tropical Plant Pathology, 33(1), 41-48. https://doi.org/10.1590/S1982-56762008000100007
Srinivas, A., Pushpavathi, B., Lakshmi, B. K. M., & Shashibhushan, V. (2017). Detection of seed borne mycoflora of sunflower (Helianthus annuus L.). The Pharma Innovation Journal, 6(9), 256-259.
Staden, R., Beal, K. F., & Bonfield, J. K. (1998). The Staden package. In S. Misener S., & S. A. Krawetz (Eds.), Bioinformatics methods and protocols (pp.115-129). Humana.
Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30(9), 1312-1313. https://doi.org/10.1093/bioinformatics/btu033
Team, R. C. (2019). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing. Retrieved November 10, 2023, from https://www.R-project.org/.[Google Scholar].
Teixeira, H., & Machado, J. D. C. (2003). Transmissibility and effect of Acremonium strictum in maize seeds. Ciênc. Agrotec., 27(5), 1045-1052. https://doi.org/10.1590/S1413-70542003000500011
Torres-Cruz, T. J., Whitaker, B. K., Proctor, R. H., Broders, K., Laraba, I., Kim, H. S., Brown, D. W., O’Donnell, K., Estrada-Rodriguez, T. L., Lee, Y., Cheong, K., Wallace, E. C., McGee, C., Kang, S., & Geiser, D. M. (2022). FUSARIUM-ID v. 3.0: an updated, downloadable resource for Fusarium species identification. Plant Disease, 106(6), PDIS-09. https://doi.org/10.1094/PDIS-09-21-2105-SR
USDA (2023). Oil Crops Yearbook. Economic Research Service, United States Department of Agriculture. Retrieved February 10, 2024, from https://www.ers.usda.gov/data-products/oil-crops-yearbook/
Veloso, J. S., Câmara, M. P., Lima, W. G., Michereff, S. J., & Doyle, V. P. (2018). Why species delimitation matters for fungal ecology: Colletotrichum diversity on wild and cultivated cashew in Brazil. Fungal Biology, 122(7), 677-691. https://doi.org/10.1016/j.funbio.2018.03.005
Walker, C., Maciel, C. G., Milanesi, P. M., Muniz, M. F. B., Mezzomo, R., & Pollet, C. S. (2016). Morphological, molecular and pathogenicity characterization of Fusarium acuminatum and Fusarium verticilioides to Cordia americana seeds. Ciência Florestal, 26(2), 463-473. https://doi.org/10.5902/1980509822747
Zhang, Y., Zhang, J., Gao, J., Zhang, G., Yu, Y., Zhou, H., & Zhao, J. (2018). The colonization process of sunflower by a green fluorescent protein-tagged isolate of Verticillium dahliae and its seed transmission. Plant Disease, 102(9), 1772-1778. https://doi.org/10.1094/PDIS-01-18-0074-RE
Zhao, L., Wei, X., Zheng, T., Gou, Y. N., Wang, J., Deng, J. X., & Li, M. (2022). Evaluation of pathogenic Fusarium spp. associated with soybean seed (Glycine max L.) in Hubei Province, China. Plant Disease, 106, 3178-3186. https://doi.org/10.1094/PDIS-12-21-2793-RE

Download PDF

Version 1

posted

You are reading this latest preprint version

Polyphasic identification of Fusarium species associated with sunflower seed-borne diseases in Brazil

Status:

Version 1

Abstract

Figures

Introduction

Material and Methods

Statistical analysis

Results

Discussion

Declarations

Acknowledgment

Conflict of interest statement

References

Supplementary Files

Status:

Version 1