Fig 1.
Germination on and penetration of the surface of mature nectarines by M. fructicola conidia and hyphae.
The nectarine’s tissues and fungal structures were stained with lactophenol blue (A, B, E and F) or Calcofluor White fluorescent stain (C and D). A-B: Clusters of non-germinated (blue) and germinated (arrow) M. fructicola conidia (c) around the guard cells of stomata (s) on the surface of mature fruit after a 4-hour incubation at 25°C. C and D: Conidia and long germ tubes (h) on the surface of mature fruit after a 16-hour incubation at 25°C. Scale bar = 50 μm. E: Long germ tubes on the surface of mature fruit after a 24-hour incubation at 25°C. F: thick ramified hyphae (rh) on the surface of mature fruit after a 24-hour incubation at 4°C.
Table 1.
Timeline of tissue changes in nectarines with a visible and a latent M. fructicola infection.
Fig 2.
Light microscopic analysis of tissues from nectarines with a visible or latent M. fructicola infection.
The nectarine's tissues were stained with Safranin O-fast Green stain and the unaffected walls of the nectarine's cells are blue and the affected nectarine’s cells, and the hyphae and conidia of M. fructicola are red: A, B and K: representative images of epidermal, subepidermal and mesocarp cells from uninoculated nectarines; C and D: signs of M. fructicola infection in areas which surround the stomata and in stomata-free areas of nectarines with a visible infection after a 24-hour incubation at 25°C. E and F: invasion and collapse of the uppermost layer of epidermal cells of nectarines with a visible infection after a 48-hour inoculation at 25°C. G and H: Collapse of epidermal cells, apparition of lysogenic cavities in the mesocarp, and extensive colonization of the deep subdermal tissues by M. fructicola of nectarines with a visible infection after a 72-hour incubation at 25°C. I and J: Colonization of the epidermis and mesocarp by M. fructicola with thin and thick hyphae, collapse and disruption of the nectarine's epidermal and mesocarpic cells, lysogenic cavities in the subepidermis, degradation of the cuticle and epidermis, and M. fructicola sporulation after a 96-hour incubation at 25°C. L: Colonization of the stomata of nectarines with a latent M. fructicola infection after a 72-hour incubation at 4°C. M: Increased colonization of the stomata of nectarines with a latent M. fructicola infection after a 144-hour incubation at 4°C. N: Colonization of the subdermal tissues by M. fructicola with the collapse of the epidermal cells was observed after 216-hour incubation at 4°C. O: Collapse of the epidermal cells and colonization of the subepidermis by M. fructicola of nectarines with a latent M. fructicola infection after a 288-hour incubation at 4°C. Scale bar = 50 μm.
Fig 3.
Ultrastructure of tissues from healthy nectarines and nectarines with a visible M. fructicola infection after a 24-hour incubation at 25°C.
A and B: The cuticle (cu), cell wall (cw) and ultrastructure of epidermal and hypodermal cells of healthy nectarines, scale bar = 5000 nm. C-E: Conidial germination (co) and germ tubes (gt) of M. fructicola on the surface of the cuticle of nectarines with a visible infection, scale bars = 2000 nm (C and D) and 500 nm (E). F and G: Intercellular hyphae (h) of M. fructicola, scale bar = 2000 nm.
Fig 4.
Ultrastructure of tissues from healthy nectarines and nectarines with a visible M. fructicola infection after a 72-hour incubation at 25°C.
A: The parenchyma of an uninfected nectarine, scale bar = 5000 nm. B, C, and F: Intracellular hyphae of M. fructicola in a mesocarpic cell of a nectarine with a visible infection, scale bars = 2000 nm (B and F) and 500 nm. D: The intracellular and intercellular hyphae of an M. fructicola in a mesocarpic cell and in the space around a mesocarpic cell of a nectarine with a visible infection, scale bar = 5000 nm. E: The intercellular hypha of M. fructicola in a mesocarpic cell of a nectarine with a visible infection, scale bar = 2000 nm.