Seeds play an important role in producing an optimum yield through healthy crop production. Healthy seeds, particularly pathogen-free seeds, are necessary for the maintenance of optimum plant populations and production.
spp. was the most frequently isolated fungal species among all isolates obtained in 2020 (70% of fungal isolates) and their average presence per sample was about 10-15%. Followed by species belonging to the genera Penicillium, Rhizopus
which were present in a slightly smaller percentage.
species at were also detected on seed vegetable pea, up to 10% of infected seeds per sample. A total of 15 representative Fusarium
isolates obtained from diseased pea seeds were examined macroscopically and microscopically. During morphological observation, seven isolates produced abundant aerial white mycelium initially and gradually turned violet with aging pigmentation on PDA (Fig 1a). After transfer to CLA (Fig 1b), these isolates formed microconidia in long chains or cohering in false heads. Slightly curved rather than straight macroconidia were formed, with a curved apical cell, mostly three to five septate, with average dimensions of 31 to 53 × 3.4 to 4.1 µm. Chlamydospores were absent. At the eight isolate noticed abundant, loosely floccose, whitish aerial mycelium with beige pigmentation (Fig 1c). On the CLA (Fig 1d), these isolates formed macroconidia with a tapered and elongated apical cell and prominent foot-shaped basal cell, which were typically four to five septate, with average dimensions of 21 to 60 × 2.8 to 4.6 µm. The isolate formed chlamydospores, but microconidia were not observed. Based on the description given by Leslie and Summerell (2006)
, cultural and morphological characteristics indicated that the seven isolates belong to F. proliferatum
and eight isolates were identified as Fusarium equiseti
(Corda) Sacc, respectively.
Fig 1: Colony of Fusarium proloferatum: (A): PDA (potato dextrose agar), (B): CLA (carnation leaf-piece agar) and F. equiseti: (C): PDA, (D): CLA.
Pathogenic fungi of the genus Fusarium
were identified in the analyzed pea seeds, but the severity of infections was generally low and the presence of this fungus did not significantly affect the quality of the seed. According to our knowledge, this is the first report of F. proliferatum
and F. equiseti
presence on pea seeds in Serbia.
Currently, the identification of members of the genus Fusarium
is based on the characteristic morphology of the colonies and microscopic characters, which include the production of multiseptal sickle conidia called macroconidia (Hue et al., 1999).
There are reports that mention the difficult recognition of Fusarium
species when macroconidia are not produced in culture (Guarro and Gene, 1995)
, because we checked our morphological results using the molecular method based on the TEF gene, whereby the presence of F. proliferatum
and F. equisets
on pea seeds was successfully detected and confirmed.
Symptoms such as discoloration, necrosis and later brown rot of the stems were observed two weeks after inoculation. The inoculated fungi were consistently isolated from the diseased plants in order to complete Koch’s postulates and proved to be the causative agent of the disease. Neither the symptoms were visible nor could the pathogen be isolated from the negative control.
All the symptoms exhibited by inoculated plants are in correlation with earlier descriptions (Chehri et al., 2011).
According to Agarwal and Sinclair (1997)
, it is essential to distinguish pathogens from other nonpathogenic fungi before establishing the inoculum thresholds for seeds to avoid a false positive assessment of seed health quality.
Molecular identification and characterisation of Fusarium spp.
The identification of four representative isolates (Ps1, Ps18, Ps19 and Ps36) based on morphological features at species level was further confirmed by molecular analyses. Molecular detection utilizing PCR and primers specific for the TEF gene successfully amplified one clear band of approximately 700 bp in all four Serbian isolates, as well as the positive control.
The amplified DNA fragment of four isolates were sequenced in both directions using the TEF-1α and deposited in the GenBank. Sequencing the gene for translation elongation factor-1 alpha (EF1α) from the two Serbian isolates Ps18 and Ps19 (MZ351883 and MZ351884, respectively) of F. equiseti
, revealed that they share 100% nt identity with Glycine max
isolates Carm34 (MH315929), Carm35 (MH315930) and Carm 36 (MH 315931) from Canada. Isolate Ps1 (MZ351881) of F. proliferatum
share 100% nt identity with ARSO-4 (KX940970) from USA, while isolate Ps36 (MZ351882) share 100% nt identity with EF10 (MN861748) from Spain.
A maximum likelihood tree (Fig 2), which was reconstructed based on the 1-α TEF sequences of different Fusarium
species selected from GenBank, shows that Ps1 and Ps36 isolates obtained in this study were grouped with the isolates previously characterized as F. proliferatum
(Acc. Nos. KF715258, KJ128964 and MK061541) while the Ps18 and Ps19 isolates were grouped with the isolates previously characterized as F. equiseti
(Acc. Nos. MH315929, MH315930 and MH 315931).
Fig 2: Phylogenetic tree (Maximum likelihood) showing the relationships among Fusarium proliferatum (Acc. Nos. MZ351881 and MZ351882) and F. equiseti (Acc. Nos. MZ351883 and MZ351884) from Pisum sativum compared with isolates of other Fusarium species, based on the sequences of elongation factor 1á (EF-1 a) gene. The four Serbian isolates are framed.
PCR is a highly effective method for identifying pathogenic fungi (Kulik et al., 2005).
This analysis is particularly useful in diagnosing seed-borne diseases which pose a threat in early stages of plant growth.
and Aoki et al., (2003, 2005)
found that the DNA sequences of the ITS region can clearly represent evolutionary links between this complex of species and the TEF-1α gene sequence always offers finer resolution and separates strains of most species of the Fusarium
complex by species. Therefore, a molecular study using TEF-1α gene was used for accurate identification of Fusarium
species in this study. Also, molecular detection based on the TEF gene of Fusarium
species could be a powerful tool in the identification of pathogenic species, giving results in a shorter period of time compared to morphological identification (Pavlovic et al., 2016).
Morphological characteristics of pathogenic isolates of Fusarium
spp., especially conidial width, were important for the selection of isolates for further research. Differences and similarities at the genetic level were confirmed by sequencing the TEF-1α region.