C.H. Beckman

A system of defence in depth provided by vascular parenchyma cells of tomato in response to vascular infection with Fusarium oxysporum f. sp. lycopersici, race 1

Abstract The penetration and colonization of xylem parenchyma cells surrounding xylem vessels of tomato infected with Fusarium oxysporum f. sp. lycopersici were examined. The primary xylem tissues of both resistant and susceptible near-isolines were found to be highly resistant to infection with only 5% and 3%, respectively, of the first tier of cells and none of the second layer of cells being invaded. The secondary xylem tissues were considerably more susceptible to infection, and here the difference between resistant and susceptible cultivars was pronounced. Secondary tissues were selected in which at least three layers of xylem parenchyma cells abutted the infected vessels. The cells in these layers were designated contact cells (those in immediate contact with the vessels) and first and second adjacent cells. The progress of infection and apparent defence responses were recorded within these three layers of cells at 3 and 6 days after inoculation. These data were later combined when it became apparent that by 3 days after inoculation a successful defence had already been established within the three cell layers, or the infection front had moved beyond them. The percentages of cells infected within these three cell layers were 52, 15 and 3, respectively, in the resistant cultivar, with a mean of 23%. In the susceptible cultivar these percentages were 90, 83, and 70, respectively, with a mean of 81%. Although similar responses, callose deposition and an apparent hypersensitive reaction were observed in both cultivars, the responses, especially that of callose deposition, appeared to be much stronger and to provide a more effective defence in the resistant cultivar than in the susceptible cultivar.

Ultrastructural responses of vessel contact cells in cotton plants resistant or susceptible to infection by Fusarium oxysporum f. sp. vasinfectum

When vascular elements of cotton plants became infected with Fusarium oxysporum f. sp. vasinfectum , contact parenchyma cells surrounding the initially infected vessels responded with changes in their ultrastructural characteristics that could be categorized into three groups. In one group, contact cells were invaded by the fungus and rapidly disintegrated within 24 h after inoculation. In the second group, the cytoplasm in contact cells degenerated within 48 h after inoculation but without apparent hyphal invasion. The evidence suggests that this cytoplasmic degeneration may represent a type of defence response. In the third group, which comprised the majority of contact cells, the cytoplasm remained uninvaded and appeared healthy after inoculation; however, it showed ultrastructural evidence of cytoplasmic reorganization and increased metabolic activity that resulted in the deposition of a complex wall apposition, as well as the development of osmiophilic droplets in the cytoplasm and their apparent secretion through the plasmalemma into the apposition layers and vessel lumens. Lateral colonization by the fungus into the vascular parenchyma cells in both cvs Seabrook Sea Island SBSI and Rowden seemed to be prevented by these cellular responses. The responses, however, were produced faster and were more pronounced in the contact cells of the resistant cultivar than in the susceptible cultivar. Thus is appears that quantitative differences in these reactions may contribute to the degree of resistance to Fusarium .

The ultrastructure of tylose formation in resistant banana following inoculation with Fusarium oxysporum f. sp. cubense

Abstract In a resistant banana cultivar tylose initials appeared within vessel lumina of roots two days after inoculation with race 1 of Fusarium oxysporum f.sp. cubense. The tyloses were formed as extensions of contact (paratracheal) parenchyma cells. By 8 days after inoculation the tyloses had enlarged to such an extent that the vessel lumina were completely occluded and walls of adjacent tyloses adhered to form a tissue that completely blocked the vessel interior. Cytological changes that occurred during their development suggest that the growth of tyloses resulted from processes common to normal cell enlargement. The formation of a “protective layer” that occurred in some contact parenchyma cells was not associated with subsequent tylose formation, but may represent a variation of the same basic process.

Ultrastructure and histochemistry of lipoidal droplets in vessel contact cells and adjacent parenchyma cells in cotton plants infected by Fusarium oxysporum f.sp. vasinfectum

Abstract Osmiophilic droplets developed in contact cells and adjacent vascular parenchyma cells of cotton accompanied by changes in cytoplasmic organization after vessel infection by Fusarium oxysporum f.sp. vasinfectum. In the early stages of the cellular response to vessel infection, the droplets were observed in dilations of ER cisternac, the membrane envelopes of mitochondria, and occasionally the membrane envelopes of plastids and nuclei. As the cellular response to vessel infection proceeded and cytoplasmic reorganization became apparent, the droplets were associated with vesicles or remained as individual droplets within the cytoplasm. The developmental sequence suggests that the osmiophilic substances in these droplets may be secreted through the plasmalemma, infused into apposition layers, and exuded into the infected vessels through pits. Sudan Black B staining and organic solvent extraction indicate that the osmiophilic droplets are lipoidal in nature. It is suggested that the lipoidal substances are terpenoids that could function as phytoalexins.

Correlated light and electron microscope studies of callose deposits in vascular parenchyma cells of tomato plants inoculated with Fusarium oxysporum f. sp. lycopersici

When vascular elements of tomato plants become infected with Fusarium oxysporum f. sp. lycopersici or root microflora, contact parenchyma cells that ensheath the vessels commonly respond by depositing a callose-containing substance along the intervening wall and especially at pit sites. These callose-containing deposits were first identified in contact parenchyma cells by light microscopy using thick sections and alkaline aniline blue staining, and then characterized ultrastructurally using TEM examination of sequential thin sections from the same cells. They could be differentiated ultrastructurally from pit membranes in both protoxylem and metaxylem tissues, and from the protective layer which has been demonstrated to occur in metaxylem contact cells as part of normal developmental and maturation processes. The callose-containing deposits often appear as electron-translucent globules with electron-opaque centres. The globules become fused into variously osmiophilic and electron-opaque aggregates, conferring a layered or marbled appearance that is distinctive to these deposits.

Growth inhibitors associated with fusarium wilt of tomato

The localization of vascular infections by a process of vascular occlusion is a primary basis for single dominant gene resistance of tomato to fusarium wilt. Failure of this response process to prevent systemic spread of fusarium in susceptible tomato plants is associated with an inhibition of tylose development, a key factor in the overall walling off process. This research was designed to detect, isolate and characterize inhibitory metabolites that accumulate during the course of the tomato/ Fusarium interaction which could inhibit tylose development. Isolation of inhibitors from chloroform extracts of systemically infected tissues was achieved by a series of successive chromatographic separations and bioassays. RFValues of inhibitors in the several solvent systems employed and their respective colour reactions with chromogenic reagents were recorded. The accumulation of a highly inhibitory compound in chloroform extracts during the early stages of Fusarium infection was determined quantitatively by colourimetric assay in a subsequent experiment. This compound, tentatively identified as rishitin, was isolated from the region of initial infection in stem vascular tissues 1–11 days after inoculation. This inhibitor accumulated rapidly in near-isolines of both resistant and susceptible tomato cultivars in 1–4 days and then continued to accumulate in susceptible, but declined in resistant, plants from 5 to 11 days. The relative phytotoxicity of this compound at low concentrations was determined by assessing its effects on the viability of cultured tomato cells. These data, together with measurements of its accumulation and determinations of its distribution in infected tissues, indicates that rishitin, a host product that accumulates in response to infection, could account for the inhibition of tylose development observed in susceptible tomato plants following infection by Fusarium. Thus we are faced with a paradox in that a substance that presumably functions in defence can, under certain circumstances, interfere with the defence process. A likely resolution of this paradox is discussed.

Enlargement and vacuolization of the cytoplasm in contact cells of resistant and susceptible tomato plants following inoculation with Fusarium oxysporum f. sp. Iycopersici, race 1*

This study deals with rapid early cytoplasmic responses that occur in contact parenchyma cells that lie immediately adjacent to vascular infections. Earlier studies have shown that such contact cells in tomato and cotton respond to vascular infections by the deposition of apposition wall layers and the synthesis of stress metabolites several hours to several days after inoculation. These responses appear to be part of a complex system of defence. The studies reported here demonstrated that pronounced cytoplasmic changes, which seemingly enable subsequent morphological changes, could be detected 60–90 min after inoculation. The changes included, first, an increased cytoplasmic width (plasmalemma to vacuolar membrane) on the side of the cell proximal to the infection, and later, increased cytoplasmic width on the distal side. No changes in the numbers and/or distribution of mitochondria were detected during this early period. The cytoplasmic response was found to be more rapid and greater in a resistant than in a near-isogenic, susceptible cultivar of tomato. Vacuolization of the cytoplasm was a conspicuous feature associated with this apparent cytoplasm distension.