Immunocytochemical localization of fusarium toxins in infected wheat spikes by Fusarium culmorum

Abstract

Two antisera raised against deoxynivalenol (DON) and 3-acetyldeoxynivalenol (3-ADON) were used to investigate the subcellular localization of the fusarium toxins, DON, 3-ADON and 15-ADON, in Fusarium culmorum infected wheat spikes and kernels by means of the immunogold labelling technique. The hyphae of the pathogen produced the toxins when they grew on the surface of the lemma and the ovary as early as 36 h after inoculation, after this incubation time the toxins were also found in the parenchyma cells. When the hyphae grew in the host tissues, more toxins were detected in the host cells, especially in the cells in close contact with the hyphae. Toxin accumulation showed a very close relationship with pathogenic changes in host cells, symptom appearance and colonization of host tissues by hyphae, suggesting that the toxins might play an important role in the disease development. The toxin labelling patterns in hyphal cells and in the infected host tissues for DON antiserum were very similar to that for 3-ADON antiserum, but the labelling density for DON was obviously higher than that for 3-ADON. In hyphal cells, the toxins were localized in the cytoplasm, mitochondria, vacuoles and the cell wall. The young hyphal cells usually showed more gold labelling in the mitochondria and the dense cytoplasm. In the host plant cells, the toxins were detected in the cytoplasm, chloroplasts, plasmalemma, cell wall and vacuoles, and sometimes gold particles were found associated with endoplasmic reticulum and ribosomes in the host cytoplasm. Four–6 days after inoculation of spikelets, hyphae reached the rachis and spread inside and outside of vascular bundles of the rachis. The toxins were present in the xylem vessels, phloem sieve tubes, paratracheal parenchyma cells and the parenchyma cells outside the vascular bundles. By 10 days after inoculation no hyphae were found in the rachis, glume, lemma or young kernels which were located three spikelets above the inoculated spikelets. However, immunolabelling of these tissues showed that toxins were present, especially in xylem vessels and phloem sieve tubes. Ten days after inoculation toxins were also detected in phloem sieve tubes of the rachis three spikelets below the inoculated spikelets. These results suggest that the toxins can be translocated upwards through the xylem vessels and phloem sieve tubes, and downwards through the phloem sieve tubes. In the infected wheat kernels, the toxins were shown not only in the hyphae, but also in the pericarp tissues, pigment strand, aleurone cells and starchy endosperm in different concentrations.