Host Cell Necrosis Research Articles

Responses to larval Taenia taeniaeformis in mice with severe combined immunodeficiency (scid).

Responses to Taenia taeniaeformis infection were studied in mice with severe combined immunodeficiency (scid), which lack functional T and B lymphocytes. In the early phase of infection, accumulation of polymorphonuclear leucocytes (PML) occurred around the larvae in the liver of scid mice and their immunocompetent counterparts, C.B-17, (a BALB/c strain, genetically resistant to this parasite). PML accumulation continued until encapsulation of developing larvae by fibroblasts (14 days p.i.), and subsequent fibrosis resulted in granuloma formation. No infiltration of eosinophils or macrophages around larvae was observed in scid mice prior to granuloma formation, while in C.B-17 mice infiltration was observed as early as 5 days p.i., when specific antibodies could not be detected in the circulation. Most larvae were destroyed by 14 days p.i. in C.B-17 mice. In scid mice the larvae survived but the host capsules (cysts) were thin and most contained blood at 42 days p.i. In these cysts, inflammatory cells were observed on the larval surface and in invaded parasite tissue. Hepatocyte coagulation necrosis adjacent to larvae was commonly found in C.B-17 mice by 5 days p.i., while it did not occur in scid mice throughout these experiments. These results suggest that in host responses to larval T. taeniaeformis, PML accumulation and encapsulation by fibrosis are T and B cell independent, while eosinophil and macrophage infiltration, as well as resistance to infection, are T and/or B cell dependent. Additionally, there may be an association between host cell necrosis around larvae and T and/or B cell responses.

Infection of and Colony Development within Leaves of Susceptible and Resistant Pearl Millet and Two Nonhosts by the Rust Fungus Puccinia Substriata Var. Indica

The rust fungus Puccinia substriata var. indica produced germ tubes, appressoria, and substomatal vesicles on seedling and mature leaves of susceptible and resistant pearl millet as well as two nonhost plants, corn and peanut. Lack of directional germ tube growth on peanut leaves appeared to affect the ability of germ tubes to successfully locate stomates. In seedling leaves of the susceptible cultivar Tift 23DB, the fungus produced an extensive system of intercellular hyphae and sporulated abundantly. In seedling leaves of the moderately resistant cultivar 86–8770, necrosis of host cells in response to fungal infection occurred by 2 days post-inoculation. Colonies gradually expanded in size and were associated with macroscopic necrotic flecks with sporulation occurring at 15% of the infection sites. In seedling leaves of the highly resistant cultivar Tift 85DB, fungal colonies were extremely limited in growth and did not sporulate. They were associated with intensely autofluorescent, necrotic host cells. Mature leaves of each cultivar were more resistant to fungal colonization than seedling leaves. Some colonies in mature leaves of Tifit 23DB were completely encompassed by necrotic cells and did not expand further. No sporulation was noted in mature leaves of 86–8770 or Tift 85DB. Fungal colonies were not successfully established in either nonhost. Thickening of plant cell walls at sites of attempted infection was noted in corn. In peanut, guard cells underneath fungal appressoria became autofluorescent.

Fungal development and host cell responses to the rust fungus Puccinia substriata var. indica in seedling and mature leaves of susceptible and resistant pearl millet

The rust fungus Puccinia substriata var. indica established a compatible relationship with host cells, characterized by large numbers of haustoria and an extensive system of intercellular hyphae, in seedling leaves of the susceptible pearl millet cultivar Tift 23DB. At some infection sites, however, necrotic host cells and papillae formed by plant cells adjacent to infection hyphae or haustorial mother cells were noted. In seedling leaves of the moderately resistant cultivar 86-8770, the initial interaction between host cells and the pathogen was quite variable and included successful haustorium formation as well as papilla deposition. Necrosis of host cells apparently developed as a gradual disorganization of the cytoplasm of invaded and surrounding host cells and occurred at all infection sites by 2 days postinoculation. In seedling leaves of the highly resistant cultivar Tift 85DB, haustoria were established at early stages of development, followed by a rapid necrosis response at 1 day postinoculation. Host cell disintegration was noted both before and after abnormalities in haustoria were observed. In mature leaves of all three cultivars, wall deposits were quite extensive at 12 h postinoculation. In addition, necrotic plant cells appeared rapidly in both susceptible and resistant cultivars. Both of these factors may have contributed to the increased resistance to fungal colonization observed in mature leaves. Key words: Puccinia substriata var. indica, pearl millet rust, host resistance, ultrastructure.

Resistant responses in juvenile seedlings of Pinus densiflora (Japanese red pine) inoculated with Endocronartium harknessii

Hypocotyls of Pinus densiflora, a species known to be resistant to western gall rust, were inoculated with Endocronartium harknessii and examined by light and electron microscopy. Host cells, when initially infected, were apparently unaffected, as were the haustoria within them. Seedlings were observed to respond to infection in two ways. In the first type of response, infected cells showed signs of necrosis by 9 days after inoculation, although infecting haustoria appeared normal. By 18 days, most cortical cells in the centre of the infected region were necrotic, as were the haustoria within them. Infected cells at the colony margin still appeared healthy, indicating that host cell necrosis lagged behind infection and only occurred after the haustorium was established. Four weeks after inoculation, a ring of suberized and lignified endodermal cells separated the infected cortex from the uninfected vascular tissue and appeared to prevent further inward growth of the fungus. The second response type involved production of encapsulations around haustoria. Encapsulations appeared to have formed after haustoria senescence and were eventually followed by host cell death.

Histological studies of the expression of the Lr9, Lr20 and Lr28 alleles for resistance to leaf rust in wheat

The development of the leaf rust fungus (Puccinia recondita f.sp. tritici) in a susceptible cultivar and three other cultivars possessing the Lr9, Lr20 and Lr28 alleles for resistance was studied by light and fluorescence microscopy. Formation of the substomatal vesicle, intercellular hypha and the first haustorial mother cell was unaffected by resistance. Lr9 and Lr28 expression was rapid, first seen as early initiation of hyphal branching at 16 h after inoculation, then reduced haustorial diameters at 19 h. Limited host cell necrosis was seen immediately afterwards. Elongation of intercellular hyphae was reduced between 20 and 24 h, and virtually ceased by about 30 h. Numbers of infection sites with a second haustorial mother cell were briefly higher at 24 h. Reduced hyphal branching and haustorial mother cell numbers were seen at 20–24 h and 36 h respectively. Lr20 expression was not seen until 36 h when reduced hyphal branching was observed, accompanied by extensive host cell necrosis. Reduced haustorial mother cell numbers were detected at 48 h. Findings suggested a secondary role for host cell necrosis in the expression of the Lr9 and Lr28 alleles. Host necrosis may play a determinant role in Lr20‐based expression.

Differences in the extent of fungal development, host cell necrosis and symptom expression during race‐cultivar interactions between Phaseolus vulgaris and Colletotrichum lindemuthianum

The cellular reactions of excised hypocotyl segments of 31 cultivars of Phaseolus vulgaris to infection by two races of Colletotrichum lindemuthianum were surveyed. Consistent differences in the extent of fungal development and host cell necrosis were used to establish six infection categories.Extreme susceptibility (category 5) involved initial formation of infection vesicles in living epidermal cells and a prolonged period of intracellular biotrophic development prior to necrotrophic growth and production of spreading lesions. Extreme resistance (category 0) involved rapid death and browning of penetrated epidermal cells, without formation of infection vesicles or a detectable biotrophic phase. Fungal growth and host necrosis were limited to single epidermal cells. Intermediate reactions (categories 1 to 4) initially resembled category 5, but after a biotrophic phase of varying duration, infected cells and some adjacent uninfected cells died and became brown, and fungal growth ceased. Symptoms ranged from flecks (small groups of dead cells) to large areas of necrosis (limited lesions). Similar intermediate reactions also occurred in normally susceptible hypocotyls which were transferred from 17 to 25°C, or which retained cotyledons.The finding that resistance is expressed at different stages of infection is discussed in relation to the regulation of cultivar specificity.

The establishment of systemic infection in leaves of oilseed rape by Leptosphaeria maculans

In a series of growth room experiments in which leaves of Brassica napus var. oleifera were inoculated with ascospores or pycnidiospores of Leptosphaeria maculans successful infections progressed through three consecutive phases. Initial establishment in the mesophyll was succeeded by a phase of intercellular exploration, when hyphal proliferation was highly variable and host cell necrosis always ensued, and then by a systemic phase when hyphae were consistently sparse. Host cells associated with the hyphal front were capable of autofluorescence, accumulation of vital stains and plasmolysis, indicating that they were viable and that the pathogen was biotrophic throughout this sequence. During either of the first two phases permanent fungistatic containment, involving the formation of vesicles by disintegration of the hyphae, often occurred. Localization at the first phase was symptomless; at the second it was signified by a lesion with a clearly defined margin.There was a negative correlation between biotrophic potential and necrotrophic potential of three pathogenic isolates, on both the moderately susceptible cultivar Primor and the resistant cultivar Jet Neuf. As leaves aged, a progressively larger proportion of infections failed to become systemic. With increasing inoculum load, symptomless localization of infection diminished, the phase of necrosis extended, and the probability of irreversible systemic development increased.

Prostacyclin and thromboxane in benign and malignant breast tumours.

6-keto-PGF1 alpha and thromboxane B2 were determined by radioimmunoassay in 37 extracts of breast carcinomata, 8 fibroadenomata, 12 sclerocystic-disease specimens and 51 normal breast tissues. More prostanoids were extracted from carcinomata than from normal specimens, fibroadenomata or sclerocystic-disease tissues (P less than 0.05). The 6-keto-PGF1 alpha/TXB2 ratio was higher in carcinomata than in normal tissues and fibroadenomata (P less than 0.05) but was not significantly different from the ratio in sclerocystic disease. The prostaglandin levels and the 6-keto-PGF1 alpha/TXB2 ratios from carcinomata did not correlate significantly with age, tumour size, differentiation, lymph node status, nuclear-cytoplasmic ratio, host cell reaction, mast cells, necrosis, elastosis, fibrosis or blood vessel density. Lower nuclear density was associated with lower 6-keto-PGF1 alpha/TXB2 ratios (P = 0.01) whereas the latter value was higher when infiltration was lower (P = 0.03). There was a positive correlation between mitotic index and the 6-keto-PGF1 alpha/TXB2 ratio (P = 0.04). Cumulation of variables revealed lower prostanoid ratios in tumours greater than 2 cm without lymph node metastasis then tumours less than 2 cm with lymph node metastasis (P = 0.04). A first follow-up (14 months) showed a higher 6-keto-PGF1 alpha/TXB2 ratio in patients who developed metastasis (P = 0.04). Our study does not confirm the hypothesis that high prostacyclin levels are a good prognostic index in breast cancer.

Effect of Aliette on Bremia lactucae on lettuce

Aliette is a systemic fungicide effective against diseases caused by lower fungi but there is some question whether it acts directly on pathogens or indirectly via host defence. Downy mildew development on lettuce cotyledons was markedly decreased by application of 100–500 μg Aliette, active ingredient (a.i.), ml −1 to roots. Sporangial germination on glass and cotyledons was prevented by direct application of 10 and 25 μg Aliette ml −1 respectively. Germ-tube growth and penetration into cotyledons were markedly decreased when 25 μg Aliette ml −1 was applied directly 2 h after sporangial inoculation. The size of secondary vesicles and growth of intercellular hyphae in cotyledons were markedly affected when 250 μg Aliette ml −1 was applied 4 h after sporangial inoculation. Hyphal growth was seen to be affected 24 h later; a low incidence of necrosis of host cells was observed after a further 24 h. The findings gave reason to believe that Aliette acted directly on the pathogen in this system.

Effects of the Srl5 allele for resistance on development of the stem rust fungus and cellular responses in wheat

The development of avirulent and virulent strains of stem rust (Puccinia graminis Pers. f.sp. tritici Eriks. & Henn.) in a susceptible wheat line and two cultivars bearing the Sr15 allele for resistance was studied, mainly by fluorescence microscopy. Formation of appressoria, substomatal vesicles, infection hyphae, and the first haustorium was unaffected by resistance. The first effect of Sr15 expression was a slower rate of haustorial mother cell formation and was first seen 48 h after inoculation. Effects on hyphal branching and colony radii followed. Necrosis of host cells was first seen at 42 h, but inspection of individual infection sites showed that necrosis did not coincide with effects on haustorial mother cells. It is possible that deterioration of host cells leading to visible host cell necrosis may be related to effects on rust development. Sr15 expression gave a mesothetic reaction, first seen microscopically 60 h after inoculation. Differences between individual infection sites in this reaction may be related to the timing of the onset of necrosis.

Peroxidase activity and induced lignification in rusted flax interactions varying in their degree of incompatibility

A histological comparison of the L9, K, P, P1, P2, and P3 genotypes in flax revealed differences in their expression of incompatibility. K and P1 were only moderately incompatible, whereas P, P2, and P3 were highly incompatible, restricting fungal development completely by the 4th day after inoculation. Host cell necrosis was delayed until day 3 in P3 and day 2 in P2; it was present at day 1 in P. Peroxidase, using guaiacol as a proton donor, was detected in P after 2 days, in K at 5 days, and in L9 after 7 days. Soluble peroxidase activity increased in the rusted P genotype from day 3 and from day 7 in K. Gel electrophoresis of infected P revealed a new peroxidase isozyme at 6 days after inoculation. Three new protein bands were also detected at this stage. In 3- and 4-day-old rusted P, peroxidase was detected in the host cell walls of tissue surrounding necrotic infection sites. It was not located in the host cell walls of the compatible L9 genotype at this stage. Lignin was detected histochemically in P after 2 days, in K after 5 days, and in L9 after 9 days. The Wiesner, Maiile, and KMnO4 tests gave equivalent results. Suberin was detected in P, K, and L9 at the same time that lignin became observable.

A quantitative histological and ultrastructural analysis of interactions between the flax rust and near-isogenic host lines varying in their degree of incompatibility

Histological differences were evident in the leaves of eight near-isogenic lines of flax infected with the rust fungus Melampsora lini. Compared with the compatible L9 and M1 interactions, fungal growth was progressively more restricted in the moderately incompatible K and M4, incompatible M and P, and highly incompatible L and M3 interactions. This restriction took place in advance of appreciable necrosis of host cells in K, M4, M, and P. At 72 h the proportion of haustoria-containing cells which were necrotic was only 10–15% in K and M4. In M at 72 h necrosis was 80% or more at infection sites with small colonies but was negligible at sites with large colonies. In P, by contrast, a similar proportion of necrosis, 40% at 72 h, was present at all infection sites. However, in L and M3 host necrosis was much more rapid and the fungus was restricted to a few host cells. An early ultrastructural event was the appearance of extensive fibrillar deposits in the initially electron-lucent extrahaustorial matrices of both the incompatible M and P and moderately incompatible M4 and K interactions. A positive reaction with silver proteinate indicated that these matrical deposits contained carbohydrate, possibly a mucopolysaccharide or glycoprotein, but they were not extracted by either cellulase, pectinase, or chitinase. The extrahaustorial membrane, surrounding the haustoria in the compatible L9 and M1 interactions and the moderately incompatible K interaction was not stained by the periodic acid – phosphotungstic acid – chromic acid (PACP) procedure believed specific for the plasmalemma. In incompatible reactions clusters of electron-dense particles sometimes replaced starch in plastids of infected host cells. This event usually coincided with the appearance of extensive matrical deposits around fungal haustoria. At the same time particles were also found in plastids in uninfected host cells immediately adjacent to infection sites, particularly in the M and P interactions. These particles were extracted from thin sections by using pullulanase followed by α-amylase, indicating that they consisted of highly branched amylopectin.

The Hypersensitive Reaction in Tobacco Leaf Tissue infiltrated with Pseudomonas pisi 5. Inhibition of RNA Synthesis in Mesophyll Cells

AbstractThe synthesis of mesophyll cell RNA in tobacco leaf tissue infiltrated with the incompatible bacterium Pseudomonas pisi was investigated by light and electron microscope autoradiography of H3‐uridine uptake.Interpretation of the light microscope quantitative data was complicated by the oscillation in levels of cytoplasmic RNA synthesis that resulted from the physical process of fluid infiltration (seen in the control tissue). Direct comparison with the control showed that the presence of bacteria resulted in a rapid decrease in the synthesis of host cell RNA. This effect was clear within the first 1½h (induction period) of the hypersensitive reaction, where it was particularly marked in the cytoplasm of palisade cells. Electron microscope autoradiography showed that the presence of bacteria did not cause complete cessation of RNA synthesis in mitochondria and chloroplasts.The early inhibition of RNA synthesis preceeded fine structural (degenerative) change, and should be regarded as one of the primary events associated with the induction of host cell necrosis.

Electron microscopy of susceptible and resistant near-isogenic (sr6/Sr6) lines of wheat infected by Puccinia graminis tritici. III. Ultrastructure of incompatible interact

The interaction between avirulent wheat stem rust and wheat mesophyll cells containing the temperature-sensitive Sr6 gene for stem rust resistance was studied by electron microscopy. Mesophyll cells that were invaded at 26 °C (conditioning compatibility) did not develop any signs of incompatibility after they were transferred to 19 °C, at which temperature incompatibility is normally expressed. In host tissue that appeared to be invaded after the change from 26 to 19 °C, the early ultrastructural symptoms of incompatibility were a more electron-dense and often perforated invaginated host plasmalemma, disruptions of the noninvaginated host plasmalemma, vacuolation of the cytoplasm, and accumulations of electron-dense material along the membranes of the vacuoles. At later stages in the development of incompatible interactions, the electron-dense accumulations along the vacuole membranes increased in size and occurred along chloroplast and mitochondrial membranes. Eventually, the entire protoplasts were electron dense and collapsed. In haustoria and haustorial mother cells, incompatibility was usually expressed by a uniform increase in electron density of the cytoplasm. In the Sr6/P6 interaction at 19 °C, host cell necrosis was not always accompanied by fungal necrosis or vice versa. In Sr5/P5 interactions, which were examined for comparison, the intracellular symptoms of incompatibility were similar to those of the Sr6/P6 interactions.

Electron microscopy of susceptible and resistant near-isogenic (sr6/Sr6) lines of wheat infected by Puccinia graminis tritici. II. Expression of incompatibility in mesophyll and epidermal cells and the effect of temperature on host–parasite interactions in these cells

Primary leaves of near-isogenic lines of Chinese Spring wheat, either with or without the temperature-sensitive resistance gene Sr6, were inoculated with an avirulent race of stem rust, maintained at 19, 26, or 28 °C, and harvested 1–4 days after inoculation. The infected tissues were examined with an electron microscope to determine the effects of these temperatures on the expression of the Sr6 gene. At 26 °C in the Sr6 line about one-half of the haustoria in mesophyll cells were disorganized or collapsed. None of the haustoria in epidermal cells showed any structural abnormality. In the susceptible (sr6) line, most haustoria were structurally normal at 26 °C whether they were in mesophyll or epidermal cells. There were no signs of disorganization or necrosis of infected host cells of either (Sr6 or sr6) line at 26 °C. At 28 °C in both lines, all haustoria in mesophyll cells were necrotic or collapsed, but those in epidermal cells were not. No host cell necrosis was observed in genotypically resistant leaves. At 19 °C, most haustoria and invaded mesophyll cells in the Sr6 line were necrotic. In the invaded epidermal cells neither haustoria nor host protoplasts were necrotic. In contrast, in a backcross line of Marquis wheat containing resistance gene Sr5 and infected with an avirulent race, both invaded mesophyll and epidermal cells were necrotic.

Histology and physiology of compatibility and incompatibility between lettuce and the downy mildew fungus, Bremia lactucae Regel

Three races of Bremia lactucae Regel and five cultivars of lettuce were investigated to determine early events which distinguish between compatible and incompatible interactions during the infection process. The time-course of the infection process was followed histologically and by the leakage of electrolytes. Infection types were graded 1 (low = incompatible), 2 and 3 (intermediate) and 4 (high = fully compatible). Low infection types were characterized by the death of host cells during penetration or 2 to 5 h after penetration, depending upon the host gene for resistance and fungal gene for avirulence. Host cells adjacent to penetrated cells were not killed. The fungus grew out of the dead, penetrated cell, and eventually died within 12 to 24 h of penetration. Intermediate infection types were characterized by large penetration pores, a delay in onset of nuclear division in the fungus, and eventual necrosis of host cells one or more days after penetration, although the fungus continued growth and sporulated sparsely. High infection types were typical of a biotrophic relationship; the fungus grew rapidly through the living host tissue and, prior to sporulation, showed little or no tendency to kill penetrated cells of the host.The cause and significance of the rapid death of host cells observed in low infection types are discussed in relation to the cause of resistance.

A histological study of interactions between avirulent races of stem rust and wheat containing resistance genes Sr5, Sr6, Sr8, or Sr22

Primary leaves of wheat (Triticum aestivum L.) with and without resistance genes Sr5, Sr6, Sr8, or Sr22 were inoculated with avirulent races of stem rust (Puccinia graminis Pers. f. sp. tritici Eriks. & E. Henn.) and examined by fluorescence microscopy. In leaves containing the Sr5 gene for resistance, both epidermal and mesophyll cells fluoresced when interacting with the fungus, indicating incompatibility. In leaves containing the Sr6 gene, interacting epidermal cells did not fluoresce and incompatibility was expressed only in mesophyll cells.When the effect of the Sr5 gene was studied in leaves with different genetic backgrounds, it was found that most colonies developed only one or two haustorial mother cells in leaves containing this gene in Prelude, Marquis, or Reliance backgrounds, when examined up to 72 h after incubation. Conversely, in leaves with the Chinese Spring background, one-third of the colonies continued to grow and they produced macroscopically visible lesions. Our observations indicated that the Chinese Spring genotype partially altered the expression of the Sr5 gene in mesophyll but not in epidermal cells. In contrast, the Sr6 gene was more effective in the Chinese Spring background than in the Prelude background.Rust development in leaves with or without the Sr8 gene was the same up to 60 h after incubation, when incompatibility in resistant plants was first detected by the appearance of fluorescing host cells. By 72 h, mean colony size in resistant leaves was smaller than that in susceptible leaves, evidently because growth of runner hyphae was inhibited. Apparently, the P8 gene for avirulence was not expressed until colonies had reached considerable size. In leaves containing the Sr22 gene for resistance, the sequence of histological events was similar to that in leaves containing Sr8, but fluorescing host cells appeared later (72 h) and colony growth was inhibited only at 96 h after incubation. In both of these interactions, fluorescing host cells developed at the periphery of colonies when incompatibility was expressed. The host-parasite interaction in cells invaded before that time remained compatible even at later stages of colony development. In leaves containing the Sr5 or the Sr8 gene, but not in those with the Sr6 gene, the growth of some colonies was inhibited although they were not associated with fluorescing host cells. Evidently, host-cell necrosis was closely associated with reduced fungal growth in interactions involving Sr6, but not in interactions involving the resistance genes Sr5 and Sr8.

Histological studies of resistance to soybean rust, Phakopsora pachyrhizi Syd

Histological comparisons were made of susceptible, resistant, and immune responses of soybean to infection by rust, Phakopsora pachyrhizi, at intervals after inoculation. In the susceptible host there was extensive hyphal growth and vigorously sporulating uredia developed. The immune hosts showed no macroscopic symptoms of infection, but at the microscopic level exhibited a hypersensitive response which was limited to a few cells. In the resistant host the hypersensitive response was not as limited, fungal development and associated host cell necrosis were more extensive, and symptoms of infection were visible macroscopically.

Histological studies on host-cell necrosis conditioned by the Sr6 gene for resistance in wheat to stem rust

Seedlings of resistant (Sr6) and susceptible (sr6) near-isogenic lines of wheat (Triticum aestivum L.) were inoculated with a race of stem rust (Puccinia graminis Pers. f. sp. tritici Eriks. & E. Henn.) that was avirulent on the line with Sr6 and they were kept at 19, 25, 26, and 27 °C. Fluorescence microscopy was used to detect autofluorescing necrotic host cells and rust colonies after these were stained with a fiuorochrome (Calcofluor White M2R New).In leaves containing the Sr6 gene, a smaller percentage of colonies grown at 25 °C had necrotic cells associated with them than those that were grown at 19 °C. The incidence of colony-associated necrosis in these leaves could be further reduced by increasing the temperature to 26 °C and 27 °C. Similarly, the number of necrotic host cells per colony decreased with an increase in temperature. Colonies in genotypically resistant leaves were usually smaller than those in genotypically susceptible leaves, but the differences in colony sizes between these two lines decreased at the higher temperatures.When infected plants containing the Sr6 gene were kept for varying times at 25 °C and then were transferred to 19 °C, there was significantly less fungal growth and more necrosis than in plants kept continuously at 25 °C. This necrosis occurred largely in those cells that were invaded after the transfer to 19 °C, when the Sr6 gene was activated.

Effect of blasticidin S, ethionine, and polyoxin D on stem rust development and host-cell necrosis in wheat near-isogenic for gene Sr6

Seedlings of resistant (Sr6) and susceptible (sr6) near-isogenic lines of wheat (Triticum aestivum L.) were inoculated with an avirulent (P6) race of stem rust (Puccinia graminis (Pers.) f.sp. tritici Eriks. & Henn.) and kept for 2 days at 26 °C where the Sr6 gene is ineffective, treated with blasticidin S, ethionine, polyoxin D, or buffer, and transferred to 19 °C where the Sr6 gene is normally effective. One and 2 days later, leaves were stained with Calcofluor and examined by fluorescence microscopy to detect autofluorescing necrotic host cells and Calcofluor-stained stem rust colonies.Blasticidin S was phytotoxic to wheat leaves at concentrations that had no effect on fungal growth during the first 2 days after treatment. At later stages, extensive host necrosis, resulting from the phytotoxicity of this antibiotic, inhibited rust development.Ethionine and polyoxin D strongly inhibited rust development at concentrations that were not phytotoxic. In genotypically resistant leaves treated with ethionine and polyoxin D there were fewer necrotic cells associated with stem rust colonies than in leaves treated with buffer. The spacial distribution of necrotic cells was consistent with the view that necrosis occurs only in cells newly invaded after the temperature was lowered to 19 °C.The observations do not support the concept that host-cell necrosis in the hypersensitive reaction conditioned by this gene results from the death of the fungus.