Morphological characterization, pathogenicity screening, and molecular identification of Fusarium spp. isolates causing post-flowering stalk rot in maize

Abstract

Post flowering stalk rot (PFSR) of maize caused by the Fusarium species complex is a serious threat to maize production worldwide. The identification of Fusarium species causing PFSR based on morphology traditionally relies on a small set of phenomic characteristics with only minor morphological variations among distinct Fusarium species. Seventy-one isolates were collected from 40 sites in five agro-climatic zones of India to assess the diversity of Fusarium spp. associated with maize crops showing symptoms of PFSR in the field. To investigate the pathogenicity of Fusarium spp. causing PFSR sixty isolates were toothpick inoculated between the first and second node at 55 days after sowing during the tassel formation stage of the crop in Kharif (Rainy season), and Rabi (Winter season) season field trials. Ten most virulent Fusarium isolates, based on the highest observed disease index, were identified by homology and phylogenetic analyses of partial sequences of the translation elongation factor 1 α (Tef-1α). Based on morphological traits such as mycelial growth patterns and mycelial pigmentation, Fusarium isolates were divided into nine clusters. The isolates were judged to be virulent based on their ability to decrease seedling vigour in in-vivo situations and high disease severity in field experiments. Pathogenicity test during the Kharif season showed 12 isolates with virulent disease symptoms with a mean severity ranging between 50 to 67 percent disease index (PDI) whereas in Rabi season, only five isolates were considered virulent, and the mean severity ranged between 52 to 67 PDI. Based on pathological characterization and molecular identification, 10 strains of Fusarium species namely, Fusarium acutatum (2/10), Fusarium verticillioides (Syn. Gibberella fujikuroi var. moniliformis) (7/10), Fusarium andiyazi (2/10) recorded the highest diseases index. All these species are part of the Fusarium fujikuroi species complex (FFSC). The distribution of virulent isolates is specific to a geographical location with a hot humid climate. Increased knowledge regarding the variability of Fusarium spp. responsible for PFSR of maize occurring across wide geographical locations of India will enable more informed decisions to be made to support the management of the disease, including screening for resistance in maize-inbred lines.

Keywords: Fusarium; PFSR; maize; molecular characterization; morphology; pathogenicity; soilborne pathogens; translation elongation factor 1 α.

FIGURE 1

Geographical locations indicating sampling sites of Fusarium spp. Isolates.

FIGURE 2

Cultural and morphological characteristics of Fusarium spp. causing PFSR in maize. For each isolate in the top row is the upper surface of the culture plate, the middle row is the lower surface, and the bottom row is a microscopic photograph of spores. A- F6; B- F27; C- F-47; D- F52; E, F- F59; G-F41; H- Raichur; I- Mysore; J- F19; K-F2; L- F13; M-F26; N-F33; O- F13; P-F14; Q- F8; R-F9; S- F58; T-F49; U-F28.

FIGURE 3

Dendrogram derived from morphological characters of 71 isolates of Fusarium spp. collected from 5 agroclimatic zones based on pigmentation, colony colour, mycelial pattern, shape and size of macro, and microconidia and number of septa in macroconidia grown on PDA medium.

FIGURE 4

Conidial morphology of Fusarium spp. (A) Sickel-shaped Macroconidia of Fusarium oxysporum (50.3 μm ± 17.5 × 1.7 μm ± 0.8) 1000X, (B). Sickle-shaped F. verticillioides (40.0 μm ± 1.0 × 2.3 μm ± 1.0) 1000X, (C) False head and monophialide conidiogenesis of F. acutatum, 400 X, and (D) Blunt ended macroconidia of F. andiyazi (18.0 μm ± 2.6 × 2.0 μm ± 1.3) 1000 X observed in the present study.

FIGURE 5

Box plot showing the distribution of germination percent (A), root length (B), shoot length (C), seedling length (D), and vigour (E) on different days after challenging maize seeds with Fusarium isolates.

FIGURE 6

Comparative lesion size of virulent (F1,F18, Raichur), Moderately virulent (D2, F10, FUG49), and less virulent (F3, FUG 16, F39). Virulent isolates caused lesions covering 2–3 nodes, moderately virulent isolates covered entire pith between nodes, and less virulent isolates a lesion restricted to the inoculated area.

FIGURE 7

Comparative virulence among Fusarium isolates observed during the in-vitro, Kharif 2020 and Rabi 2020-21 season. Values for histograms sharing the same letter label are not significantly different (P > 0.05).

FIGURE 8

Box plot showing disease severity levels of Fusarium spp. isolates during (A) Kharif 2020 and (B) Rabi 2020-21; Within each box horizontal black line denote median values.

FIGURE 9

Phylogenetic analysis of Fusarium spp. virulent strains based on translation elongation factor (Tef-1α) sequences by MEGA X software using neighbour joining method with 1,000 bootstrap replication. Accessions in bold are strains characterized in the present experiment. Reference sequences were obtained from Crous et al. (2021). Two reference strains for each species are: Fusarium verticillioides (Reference species: MW401977 CBS 117.28 strain, MW402080 CBS 141.59 strain), F. andiyazi (Reference species: MN533989 CBS 119856 strain, MN193854 NRRL 31727 strain), and F. acutatum (Reference species: MW402124 CBS 401.97 strain, MW402125 CBS 402.97 strain). The Alternaria burnsii isolate Alt-MP6 (Sequence ID: ON993391) was used as an outgroup. Fusarium verticillioides; Fusarium andiyazi; Fusarium acutatum.