First Report of Fusarium proliferatum Infection in Pods of Four-Seeded Vetch and Its Relationships with Plants

Abstract

Vicia species are of great value in ecological restoration, soil improvement, and the development of a forage resource. In 2024, a novel pod disease affecting four-seeded vetches (Vicia tetrasperma) emerged in Rongchang District, China, leading to severe yield loss. After obtaining the main pathogenic strain, FVS1, through the tissue isolation method, which was verified according to Koch's postulates, and by combining morphological characteristics with multigene phylogenetic analysis, FVS1 was identified as Fusarium proliferatum. The biological properties indicated that the most suitable culture medium of the fungus was oatmeal agar (OA), with the optimum growth temperature 25 °C and the lethal temperature being 35 °C. FVS1 exhibited insensitivity within a pH range of 7 to 9, as well as high adaptability to variations in light duration. To elucidate the physiological and biochemical changes in four-seeded vetches in response to FVS1 infection, non-targeted metabolomics analysis identified 379 differential metabolites, mainly comprising organic acids and derivatives, lipids and lipid-like molecules, and phenylpropanoids and polyketides. The results demonstrated that F. proliferatum primarily induced the disease by influencing alterations in the secondary metabolites associated with amino acid metabolism, lipid metabolism, and flavonoid biosynthesis. Four-seeded vetches improved tolerance to the fungus by accumulating histidine, aspartic acid, arginosuccinate, ethanolamine, glycerophosphocholine, naringenin, and catechin. Trichoderma harzianum (M3) had the best control effectiveness, and the inhibition rate was 60.68%. This study, for the first time, revealed that F. proliferatum caused a pod disease in four-seeded vetches. We analyzed the mechanism of plant-pathogen interaction and screened potential biocontrol strains, providing a theoretical basis for regional disease management.

Keywords: Fusarium proliferatum; Vicia tetrasperma; biological characteristics; biological control; metabolomics analysis.

Figure A1

The morphological characteristics of four biocontrol strain-confronted cultures with FVS1 on PDA for 3 d and 7 d.

Figure A2

This histogram shows the average colony diameter of different biocontrol strain-confronted cultures with the FVS1 pathogen on PDA for 3 d and 7 d, and the line graph shows the inhibition rate. The same lowercase letters mean the difference is not significant (p > 0.05); different lowercase letters indicate significant differences (p < 0.05).

Figure 1

Infected plants in the field.

Figure 2

Symptoms of the pods of four-seeded vetches inoculated with FVS1 and healthy controls: (a–c) FVS1 was inoculated back onto the detached pods; (d–f) healthy, detached pods; (g) FVS1 was inoculated back to the four-seeded vetch plant; (h) healthy four-seeded vetch plant; (i,k,m) FVS1 was inoculated back to the pods on intact plant; (j,l,n) healthy pods on intact plant.

Figure 3

The morphology of FVS1 grown on potato dextrose agar (PDA): (a,b) the front and back morphology of FVS1 cultured for 7 d; (c–e) microconidia and macroconidia; (f,g) conidiophores and conidia; (h,i) mycelia. Gauge = 10 µM.

Figure 4

A phylogenetic tree of FVS1, constructed via the neighbor-joining method, based on p-distance and the analyses of multiple gene sequences (BT, EF).

Figure 5

The morphological characteristics of FVS1 cultured for 7 d and the average colony diameter cultured for 3 d and 7 d under different conditions: (a,e) different media; (b,f) different temperature conditions; (c,g) different light durations; (d,h) different pH conditions. The same lowercase letters mean that the difference is not significant (p > 0.05); different lowercase letters indicate significant differences (p < 0.05).

Figure 6

Metabolomics analysis of diseased and healthy pods of four-seeded vetches: (a) principal component analysis (PCA) of diseased and healthy pods of four-seed vetches; (b) orthogonal partial least squares discriminant analysis (OPLS-DA) of diseased and healthy pods of four-seed vetches; (c) OPLS-DA permutation test diagram; (d) volcanic plots (the red graph shows significantly up-regulated differential metabolites; the blue graph shows significantly down-regulated differential metabolites; and the gray graph shows unimportant differential metabolites); (e) differential metabolite classification 3D pie chart; (f) bubble chart. The x-axis represents pathway impacts, the y-axis represents pathway enrichment, and the color of the circle from red to yellow indicates a smaller p-value (p < 0.05). The size of the circle indicates the enrichment ratio.

Figure 7

Heat maps and a pathway map: (a–c) heat maps of differential metabolites of organic acids and derivatives, lipids and lipid-like molecules, and phenylpropanes and polyketides; (d) pathway map. The blue boxes are used to denote metabolic pathways, the red boxes represent significantly up-regulated metabolites, and the green boxes indicate significantly down-regulated metabolites.