Evaluation and management of fungal-infected carrot seeds

Carrot (Daucus carota L.), which is one of the 10 most important vegetable crops worldwide, is an edible root vegetable desired for its taste as well as its medicinal uses. However, a fungus isolated from carrot seeds was observed to substantially decrease the germination rate. The isolate was identified as Alternaria alternata based on morphological and molecular characteristics as well as a phylogenetic tree. The maximum seed infection rate of selected carrot cultivars was approximately 60%, with the main infection site just underneath the seed shell. Additionally, the germination rate of infected seeds decreased by 28.7%. However, the seed infection rate varied among the examined carrot cultivars. Regarding the effects of chemical fungicides, the optimal treatment involved immersing seeds in amistar top suspension concentrate (SC) (effective concentration of 0.65 g/L) for 6 h, which effectively killed the fungi inside the carrot seeds. The results of this study provide a theoretical basis for the development of efficient methods for preventing the infection of carrot seeds by specific fungi and increasing the germination rate and vigour index.

Scientific RepoRtS | (2020) 10:10808 | https://doi.org/10.1038/s41598-020-67907-5 www.nature.com/scientificreports/ The aim of the current study was to isolate the fungi associated with carrot seeds, evaluate the efficacy of chemical fungicides against the detected fungi, and optimize the application of fungicides to maintain a relatively high germination rate and vigour index.

Materials and methods
Isolation and identification of fungi infecting carrot seeds. The tested carrot seeds (cultivars Kaixinmintu and Berlin) were purchased from a commercial market in Shuangyashan, Heilongjiang province, China. The seeds (30 seeds/cultivar) were surface-sterilized with 0.5% NaOCl for 5 min, rinsed three times with sterilized distilled water, placed on potato dextrose agar (PDA), and incubated at 26 °C. Single spores were obtained from the fungal cultures for morphological and molecular analyses as previously described 16 . The fungi were identified based on morphological characteristics as described in published methods 17,18,19 . To further identify the fungi, genomic DNA was extracted from the isolates with the Fungal Genomic DNA Kit (Tiangen, Beijing, China), after which it was amplified by PCR with the universal fungal primers ITS1 and ITS4 20 and EF1-728F/ EF1-986R 21 . The PCR amplification was conducted in a 50 µl solution consisting of 25 μl Taq mixture (Promega, Madison, WI, USA), 2 µl ITS1 primer (10 μM), 2 µl ITS4 primer (10 μM), 2.0 µl DNA template, and 19 µl ddH 2 O. The PCR was completed with the T100™ thermocycler (Bio-Rad Laboratories Inc, CA) and the following program: 94 °C for 5 min; 36 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72°C for 1.5 min; 72 °C for 10 min. The amplicons were purified and sequenced by Shanghai Biological Engineering Co., Ltd. (Shanghai, China).
Effects of fungal infections on carrot seed germination. Carrot seeds (cv. Hanhong) were collected to analyse the impact of fungal infections on germination. amistar top SC (325 g/L) was diluted to 0.65 g/L (effective concentration), after which carrot seeds were immersed in the diluted fungicide solution for 8 h to kill all of the fungi within the seeds. Control seeds were treated with sterile water. All seeds were then air-dried and analysed. The seed infection and germination rates were determined by culturing on PDA. Specifically, the seeds were sterilized, after which 30 seeds per treatment were placed on PDA medium and incubated at 25 °C for 5 days. The appearance of the resulting fungal colonies was recorded. This analysis was completed with three replicates for a total of 90 seeds, which were used to calculate the seed infection and germination rates with the following equations: Carrot seed parts infected by fungi. Carrot seeds (cultivar Hanhong) were surface-sterilized with three replicates for a total of 90 seeds. The 90 seeds were cut in the middle with sterilized scissors and the halved seeds were placed on PDA medium. Additionally, 90 intact seeds were placed on PDA medium. After a 5-day incubation at 25 °C, the appearance of the fungal colonies was recorded. The seed infection rate was calculated to evaluate the seed parts infected by fungi.
The tested carrot seeds were surface-sterilized. For each carrot cultivar, 30 seeds per cultivar were placed on PDA medium, with three replicates for a total of 90 seeds per cultivar. The seeds were incubated at 26 °C for 5 days, after which the seed infection rate was calculated with the formula provided above and compared among the 10 cultivars. The chemical fungicides were diluted for an effective concentration of 0.65 g/L. Carrot seeds (180 seeds/ fungicide) were immersed in the diluted solutions or sterile water (control) for 6 h. After air-drying the seeds, 90 of them were added to PDA medium to assess the effects of the fungicides on the seed infection rate. This analysis was completed with three replicates for a total of 90 seeds. The remaining 90 seeds were added to pots containing sterilized soil, with 10 seeds per pot and nine pots per treatment. The seeds were covered with 1 cm of soil. After a 15-day incubation in the greenhouse, the germination rate, seedling height, and fresh weight were determined. The seed infection rate was calculated with the formula provided above. The following formula was used to calculate the control effect:

Results
Isolation and identification of fungi infecting carrot seeds. Ten fungi were isolated from cultivars Kaixin666 and Berlin and then subcultured by transferring hyphal tips. Single-microconidium isolates were generated from the fungal cultures as previously described 16 and the morphological characteristics of the isolates were analysed. Colonies on PDA medium consisted of dark grey mycelium (Fig. 1). Conidia produced after a 4-day incubation at 26 °C in darkness were pale brown to dark brown, straight or flexuous, obclavate to obpyriform or ellipsoid, and had a short conical beak at the tip or were beakless. The spores had a smooth or verrucose surface and were 3.6-11.5 μm × 6.3-35.2 μm, with 0-4 transverse septa and 0-1 longitudinal septa. Ten fungal isolates had identical morphological characteristics and were identified as Alternaria alternata 19 .
A combined tree based on the ITS, TEF-1ɑ and RPB2 sequences indicated that Cbailin and Akaixin were A. alternata (Fig. 2). The sequences of the Cbailin and Akaixin amplicons were deposited in the GenBank database (accession numbers MN337233 and MK332248 for ITS, and MT178330 and MT178329 for TEF-1ɑ, and MT593329 and MT593330 for RPB2, respectively). To the best of our knowledge, this is the first report of the isolation of A. alternata from carrot seeds.
Effects of fungal infections on carrot seed germination. The carrot seeds treated to kill all fungi germinated better than the control carrot seeds (Table 1). Specifically, the mean germination rate of seeds lacking A. alternata was 28.7% higher than that of control seeds infected by fungi.
Detection of carrot seed parts infected by fungi. An analysis of the carrot seed parts infected by fungi revealed the seed infection rates of the whole seed and the cut seed were both 53.3% (Fig. 3). The lack of significant difference (P < 0.05) between the seed infection rates suggested that the fungi were just underneath the seed shell (i.e., internally seed-borne and extra-embryonic).  Seed treatments with various fungicides. The treatments with nine chemical fungicides resulted in considerable variability in the seed infection rate, germination, and seedling growth (Table 3). For example, amistar top SC killed all of the fungi within carrot seeds (100% control of fungal infection), whereas captan, procymidone, and azoxystrobin were less effective (40% control of fungal infection). Moreover, boscalid, difenoconazole, carbendazim, benzofuramide, and fipronil were relatively ineffective for controlling fungal infections   Table 3. Effects of seed treatments with nine chemical fungicides on carrot seed infection rates, germination, and seedling growth.

Fresh weight (g)
Azoxystrobin 250 g/L 30.0 ± 6.8b 50. www.nature.com/scientificreports/ of seeds. On the basis of the treatment effects on seed germination and seedling growth, amistar top SC was considered to be the most appropriate chemical fungicide.

Seed treatments with varying amistar top SC concentrations. The three tested amistar top SC
concentrations were inhibitory to the fungal infection of carrot seeds ( Table 4). The 0.65 g/L treatment was the most effective (99.2% control of fungal infection). Additionally, seed germination and seedling growth were not inhibited.

Effects of varying amistar top SC seed treatment times. The four tested amistar top SC treatment
times inhibited the infection of seeds by fungi, with obvious differences among the analysed times (Table 5). Specifically, the 6-h and 8-h treatment times were the most effective, with no significant difference between these two time-points. However, plant height was significantly lower after the 8-h treatment. The 6-h seed treatment with amistar top SC was the most appropriate based on the overall performance.

Discussion
Carrots are an important vegetable grown worldwide and represent a source of carotenoids in the human diet 23 . However, the emergence rate of some carrot cultivars was relatively low. The decreased germination rate was due to an infection of the seeds by A. alternata. The genus Alternaria Nees includes imperfect fungal species that are cosmopolitan and economically important. In the current study, the germination rate of seeds not infected with A. alternata was 28.7% higher than that of the control seeds infected with fungi. Thus, screening for suitable fungicides and optimizing their applications are important. Seeds are critical for viable crop production. Pathogen-free seeds are essential for generating healthy plant populations and a good harvest 7 . In many crops, fungal infections are responsible for low-quality seeds 24 . Additionally, the presence of seed-borne pathogenic fungi in beans results in decreased germination, emergence, growth, and yield 25 . This is consistent with our finding that the highest carrot seed infection rate was approximately 60%. Moreover, seed infection rates differed among the tested carrot cultivars. Selecting carrot cultivars uninfected by fungi represents a good agronomic practice for minimizing the chances of fungal infections.
Microorganisms associated with seeds may be pathogens, weak parasites, or saprophytes. They may be present within or on the surface of seeds and may infect seeds via exposures to contaminated sclerotia, galls, fungal bodies, infected plant parts, and soil particles 6 . Fungal pathogens may be externally or internally seed-borne, extra-or intra-embryonic, or associated with seeds as contaminants 26 . To clarify which carrot seed parts are infected by fungi, the seed infection rates were calculated for surface-sterilized whole and cut seeds. Our results suggest the primary fungal infection site of carrot seeds is just underneath the seed shell (internally seed-borne and extra-embryonic). However, a more precise localization of the fungi is necessary.
Seeds infected by fungi influence the germination, overall health, and final crop stand under field conditions 7 . Seed-borne as well as seed-associated fungal infections can be effectively inhibited if the seeds are treated with fungicides before sowing 6 . The application of chemical fungicides can completely control fungal infections, but it can be costly and harmful to human health and the environmentyyyyy 6 . To prevent the undesirable effects of fungicides on carrots, we systematically screened for effective fungicides and examined the effects of their application. The optimal seed treatment involved a 6-h immersion in amistar top SC (effective concentration of 0.65 g/L), which killed all of the fungi infecting the seeds, with no deleterious effects on the seeds or seedlings.
The results of this study provide a theoretical basis for the development of effective methods for controlling the fungal infections of carrot seeds, thereby increasing the germination rate and vigour index.