Symptomless Tissues Research Articles

Fungal Species Composition in Maize Stalks in Relation to European Corn Borer Injury and Transgenic Insect Protection.

The maize stalk rot complex is comprised of several fungal pathogens, including Gibberella zeae, Colletotrichum graminicola, Stenocarpella maydis, and several members of the genus Fusarium. The European corn borer (ECB) (Ostrinia nubilalis) can contribute to stalk rot development by creating entry wounds and by serving as a vector of some stalk rot pathogens, particularly Fusarium verticillioides. Transgenic insect protection of maize hybrids with insecticidal proteins derived from Bacillus thuringiensis greatly reduces ECB injury and may therefore alter the species composition and diversity of the stalk rot complex. Field experiments were conducted in 1998, 1999, and 2000 to compare the species composition and diversity of fungi infecting stalks of Bt and non-Bt maize hybrids. Hybrids representing five Bt types (or "events") and their near-isogenic non-Bt counterparts were subjected to manual and natural infestations with ECB larvae. Stalk tissue samples were cultured to determine fungal species composition. At least one species was isolated from nearly every stalk and from both diseased and symptomless tissues. G. zeae was the most common species in 1998 and 1999, but C. graminicola was most common in 2000. The mean proportions of stalks infected with F. verticillioides, F. proliferatum, and F. subglutinans were significantly lower in Bt hybrids than in non-Bt hybrids in 2 of the 3 years. Conversely, the mean proportion of stalks infected with G. zeae was higher in some Bt hybrids than their non-Bt counterparts in two of the three years. F. verticillioides was more likely to be isolated from ECB-injured tissue, whereas G. zeae and C. graminicola were more likely to be isolated from tissue not associated with ECB injury. The overall species diversity of the stalk rot complex was lower in some Bt hybrids compared with their non-Bt counterparts in 1998 and 1999. ECB activity appeared to alter fungal species composition in stalks, reflecting the association between ECB injury and specific stalk rot pathogens, particularly F. verticillioides. The species composition of fungi infecting stalks of Bt hybrids differed from that of non-Bt hybrids, but the implications of this result are not yet clear.

Evaluation of resistance to Phytophthora cinnamomi in seed‐grown trees and clonal lines of Eucalyptus marginata inoculated in lateral branches and roots

Seed‐grown trees and six clonal lines of 3·5–4·5‐year‐old Eucalyptus marginata (jarrah) growing in a rehabilitated bauxite mine site in the jarrah forest were underbark‐inoculated on lateral branches (1995) or simultaneously on lateral branches and lateral roots (1996) with isolates of Phytophthora cinnamomi in late autumn. Individual seedlings from which the clonal lines were derived had previously been assessed as either resistant (RR) or susceptible (SS) to P. cinnamomi. At harvest, the acropetal lesion and colonization lengths were measured. Overall, the length of colonization in roots and branches was more consistent as a measure of resistance than lesion length, because colonization length recorded the recovery of P. cinnamomi from macroscopically symptomless tissue ahead of the lesion which, on some occasions, was up to 6 cm. In both trials, one RR clonal line was able to contain the P. cinnamomi isolates consistently, as determined by small lesion and colonization lengths in branches and roots. In contrast, the remaining two RR clonal lines used in both trials were no different from the SS line in their ability to contain lesions or colonization. These latter two RR lines may therefore not be suitable for use in rehabilitation of P. cinnamomi‐infested areas. Differences in lesion and colonization lengths among P. cinnamomi isolates occurred only in the 1995 trial. Colonization and lesion lengths in branches were up to eight times greater in 1996 than in 1995, but the relative rankings of clonal lines were consistent between trials. Although colonization was always greater in branches than roots, the relative rankings of the lines were similar between branch and root inoculations. Branch inoculations are a valid option for testing the resistance and susceptibility of young jarrah trees to P. cinnamomi.

The detection of tuber necrotic isolates of Potato virus Y, and the accurate discrimination of PVY O, PVY N and PVY C strains using RT-PCR

Potato tuber necrotic ringspot disease (PTNRD) is a damaging disease of potatoes, causing unsightly necrotic rings on the surface of tubers. The causal agent is thought to be tuber necrotic isolates of Potato virus Y, known as PVY NTN. The disease spoils tubers for processing and table use, and the lack of a diagnostic method makes control especially difficult. The development of an RT-PCR assay for the reliable detection of PVY NTN and discrimination of all the main strains of PVY (PVY O, PVY N and PVY C) is described. An assay was developed, exploiting a recombination site in the coat protein of PVY NTN, allowing more reliable diagnosis of these isolates. Although the conserved nucleotide differences observed between the strains was very small, competitive PCR and mutagenically separated PCR were both employed in the development of a robust assay. The assay was found to be more reliable than the most commonly used RT-PCR method, and should prove to be an important tool in the confirmation of symptoms and for the detection of PVY NTN in symptomless tissue, in disease surveys and seed health schemes.

Latent Infection of Austrian and Scots Pine Tissues by Sphaeropsis sapinea.

This study documents latent infection of Austrian and Scots pine tissues by Sphaeropsis sapinea, the causal agent of Sphaeropsis tip blight disease. Symptomless shoots from diseased or apparently healthy Austrian and Scots pine trees were cultured and analyzed for the presence of S. sapinea. Nearly half of the sampled Austrian and Scots pine trees that appeared to be completely healthy had detectable latent S. sapinea infections. More than half of the symptomless shoots on visibly diseased Austrian and Scots pine trees were also latently infected with S. sapinea. S. sapinea was isolated from symptomless shoot stems, needles, buds, immature cones, and male flowers. In symptomless infected shoots, the fungus was primarily associated with the bark and phloem tissues. Six isolates of S. sapinea from symptomless Austrian and Scots pine shoot tissues were pathogenic on Austrian pine seedlings in the greenhouse. The presence of S. sapinea in symptomless tissues of tip blight-diseased trees has important implications for disease management.

Spathiphyllum sp.: A New Natural Host of Impatiens necrotic spot virus

In September 1999, several Spathiphyllum plants grown in a greenhouse in Tuscany (Italy) showed leaf symptoms in the form of concentric chlorotic ringspots, line patterns, and irregular chlorotic blotches. These symptoms developed into localized necrosis. Crude sap of tissues showing symptoms was mechanically inoculated to young symptomless Spathiphyllum plants and to Nicotiana benthamiana and N. clevelandii. Samples drawn from symptomatic and symptomless tissues of naturally or artificially infected Spathiphyllum and Nicotiana plants were tested for the presence of Alfalfa mosaic virus (AMV), Arabis mosaic virus (ArMV), Cucumber mosaic virus (CMV), Dasheen mosaic virus (DsMV), Impatiens necrotic spot virus (INSV), Potato X virus (PVX), Potato Y virus (PVY), Tobacco mosaic virus (TMV), and Tomato spotted wilt virus (TSWV) by double-antibody sandwich enzyme-linked immunosorbent assay carried out with commercial antisera. The symptomatic tissues obtained from Spathiphyllum and Nicotiana plants gave a positive reaction only for INSV. The symptomless samples obtained from various parts of the infected Spathiphyllum plants gave a negative reaction, even after 1 year from the appearance of localized necrosis, suggesting a non-systemic infection in this new host. This is the first report of infection of Spathiphyllum sp. by INSV.

Inflammation in Chronic Venous Insufficiency: Is the Problem Insurmountable?

One of the hallmarks of venous insufficiency is an elevated venous pressure. While a number of mechanisms have been proposed for vascular and parenchymal cell damage following venous pressure elevation, such as white cell infiltration, a key question remains as to what degree venous occlusion and flow interruption per se may constitute a risk factor in venous disease. To gain an insight into this mechanism, we examined the effect of venous occlusion followed by reperfusion. A draining venule (circa 50 µm) in the rat mesentery was occluded with a micropipette (1 h) followed by reperfusion (1 h). The procedure serves to raise the microvascular pressure to about 31 mm Hg during the occlusion while the flow is completely stopped in the local venous and capillary network. Parenchymal cell death in the mesentery was monitored by propidium iodide (PI) labeling. The number of PI-positive cells significantly increased predominantly during reperfusion. A 1-week treatment with a micronized purified flavonoid fraction (100 mg/kg/day) served to significantly reduce parenchymal cell death as well as leukocyte rolling, adhesion to postcapillary venule, and migration into the tissue both during occlusion and reperfusion. The results indicate, that even in an initially symptomless tissue, flow reduction combined with microvascular pressure elevation during venous occlusion results in tissue damage not only during reperfusion (as in arterial occlusion) but also during occlusion.

The infection of non-wounded and wounded periderm tissue at the lower stem of Eucalyptus marginata by zoospores of Phytophthora cinnamomi, in a rehabilitated bauxite mine

Artificially introduced zoospores of Phytophthora cinnamomi were shown to invade non-wounded and deliberately wounded periderm at the lower stem and collar of one-year-old seedlings of Eucalyptus marginata (jarrah) growing in a rehabilitated bauxite mine, during late autumn/early winter. Lesions were not produced in the majority of seedlings despite the demonstrated presence of the pathogen in the symptomless stem tissue.

Hrp Mutant of Pseudomonas syringae pv phaseolicola Induces Cell Wall Alterations but Not Membrane Damage Leading to the Hypersensitive Reaction in Lettuce.

Both wild-type (S21-WT) and hrpD- (S21-533) strains of Pseudomonas syringae pv phaseolicola induced the formation of large paramural papillae in lettuce (Lactuca sativa) mesophyll cells adjacent to bacterial colonies. Localized alterations to the plant cell wall included deposition of hydroxyproline-rich glycoproteins, phe-nolics, and callose, and were associated with proliferation of the endoplasmic reticulum and multivesicular bodies. Tissue collapse during the hypersensitive reaction caused by S21-WT was associated with electrolyte leakage and rapid accumulation of the phy-toalexin lettucenin A, both of which followed membrane damage indicated by the failure of mesophyll cells to plasmolyze. A few cells lost the ability to plasmolyze after inoculation with S21-533, and low levels of lettucenin A were recorded, but neither leakage of electrolytes nor tissue collapse were detected. Dysfunction of the plasma membrane in cells adjacent to colonies of S21-WT led to extensive vacuolation of the cytoplasm, organelle disruption, and cytoplasmic collapse[mdash]changes unlike those occurring in cells undergoing apoptosis. Strain S21-533 remained viable within symptomless tissue, whereas cells of S21-WT were killed as a consequence of the hypersensitive reaction. Our observations emphasize the subtle coordination of the plant's response occurring at the subcellular level.

Detection and Distribution of Sweetpotato Feathery Mottle Virus in Sweetpotato by In Vitro-Transcribed RNA Probes (Riboprobes), Membrane Immunobinding Assay, and Direct Blotting

An in vitro-transcribed RNA probe (riboprobe) system was developed to detect sweetpotato feathery mottle virus (SPFMV) in infected plants. The essential components of the system include selection of an SPFMV cDNA clone that reacts with all known strains of SPFMV, optimization of assay procedures, and modified hybridization conditions. Additionally, a direct blotting technique on nitrocellulose membrane was developed to detect SPFMV by either riboprobe or membrane immunobinding assay (MIBA). The riboprobe system provided greater sensitivity of detection of virus in symptomless tissues than MIBA (...)

Association between Symptom Development and Inhibition of Ornithine Carbamoyltransferase in Bean Leaves Treated with Phaseolotoxin

The chlorosis symptom that characterizes the halo blight disease of Phaseolus vulgaris L. is caused by phaseolotoxin produced by the plant pathogenic bacterium Pseudomonas syringae pv phaseolicola. Phaseolotoxin is hydrolyzed by plant peptidases to N(delta)(N'-sulpho-diaminophosphinyl) -l-ornithine which also causes chlorosis and is reported to be an irreversible inhibitor of ornithine carbamoyltransferase (OCTase). We have examined the hypothesis that inhibition of OCTase is the primary action of phaseolotoxin that leads to chlorosis.Chlorotic spots appeared on the primary leaves of P. vulgaris seedlings during the 2 days following leaf prick application of a minimum of 30 picomole phaseolotoxin. OCTase in extracts of the lesions was reduced to 20%, or less, of the activity in controls. Four hours after the application of phaseolotoxin the concentration of free ornithine increased more than 2-fold. Other amino acids, especially glutamine and asparagine-but not arginine-increased later. Chlorophyll remained at a constant level in the phaseolotoxin-treated tissue and the appearance of chlorosis was due to the increase in chlorophyll in the surrounding unaffected tissue.Clear halo symptoms developed only on primary leaves of the youngest seedlings (treated 6-7 days after germination). Lesions did not develop on primary leaves of seedlings more than 14 days old, in which the chlorophyll concentration had reached a maximum. OCTase also was inhibited in the symptomless tissue from older leaves treated with phaseolotoxin, but there was no accumulation of amino acids, including ornithine. A single appliction of 200 nanomoles arginine resulted in the complete regreening of the chlorosis caused by phaseolotoxin. Soluble protein was lower in the chlorotic tissue than in the controls, but increased to greater than the control value following the appliction of arginine. These results suggest that phaseolotoxin-induced chlorosis results from reduced chlorophyll synthesis that is associated with arginine-starvation in the tissue where OCTase is inhibited.

Cucumber mosaic virus-associated RNA 5: VII. Double-stranded form accumulation and disease attenuation in tobacco

The double-stranded (ds) form of cucumber mosaic virus (CMV)-associated RNA 5 (CARNA 5), which accumulates in unusually large proportions in CMV-infected Xanthi nc tobacco plants, could be identified directly in buffer extracts of these plants via its reaction with poly(I):poly(C) or poly(A):poly(U) antisera in two-dimensional immunodiffusion tests. After infiltration of CMV-infected tobacco tissues with 4′-aminomethyltrioxalen, followed by irradiation with long-wavelength ultraviolet light, a certain proportion of the dsCARNA 5 was recovered in a crosslinked state. This showed that in all probability a major proportion of the accumulated (+) and (−) strands of CARNA 5 occurs intracellularly in ds form and that dsCARNA 5 is not an artifact of phenol extraction. In differential temperature inoculated tobacco leaves, in which virus multiplies semisynchronously, dsCARNA 5 continued to accumulate after the biological activities attributable to virus and CARNA 5 were declining steeply. In tobacco plants systemically infected with CMV strain P6, which produced very distinct symptoms in individual leaves as well as the characteristic alteration of layers of symptomatic and symptomless leaves, dsCARNA 5 was usually detected in symptomless tissues but virus was not. Also, in other tissues and organs of such plants the presence of dsCARNA 5 was generally inversely correlated with that of virus. In all likelihood, the accumulation of dsCARNA 5 is the manifestation of a mechanism which regulates disease expression and resistance in CMV-infected plants.

Histopathology and histochemistry of sugar beet seedlings resistant and susceptible to Aphanomyces cochlioides

Hyphae were observed within diseased cortical tissue of hypocotyl lesions of both the susceptible and the resistant varieties but were not detected in symptomless tissues in advance of lesion margins. Hyphae ramified extensively intercellularly and, to a lesser extent, intracellularly. Only in severely diseased beets were hyphae found within the stele. Protoplasts of cortical cells in proximity to hyphae appeared to lose turgor early in pathogenesis; then the cells collapsed and cortical tissue was apparently macerated. Histochemically, cell wall constituents (pectin, hemicellulose, non-cellulosic polysaccharides, lignin, suberin, and cutin) were little changed in diseased beets as compared with healthy beets. Peroxidase and (at times) phenolase activity increased and phenols and quinones accumulated in the stele of diseased plants (adjacent to lesions) to levels above those found in healthy plants. Possibly these phenols and quinones may inhibit colonization of stelar tissues. No differences in accumulation or localization of these substances in the resistant as compared with the susceptible beet varieties were detected by histochemical methods.

Induced resistance in tobacco leaves: The growth of Pseudomonas solanacearum in protected tissues

Tobacco leaves develop systemic, nonspecific resistance to a wide variety of pathogens following infiltration with heat-killed cells of Pseudomonas solanacearum. The protective response was detected by challenge inoculations with a compatible strain of this bacterium at 10 9 cells/ml, carried out at0, 2, 4, 8 and 24 h after infiltration with heat-killed cells. When populations of bacteria in infiltrated tissues were determined, protection was detectable after 2 h as a lengthening of the lag phase, although growth rates at log phase remained basically unaltered. As the period between infiltration with heat-killed cells and challenge increased, the initial population introduced decreased rapidly and there was a concomitant reduction in the growth rate established after a protracted lag period. In fully protected leaves (24 h after infiltration) bacterial numbers decreased rapidly immediately following inoculation and populations leveled off at approximately 10 7 cells/ml, but tissues remained symptomless. Similar results were obtained at lower inoculum levels (10 6 cells/ml, bxcept that, after a sharp initial reduction in the number of cells and a protracted lag, populations increased and then stabilized at approximately 10 7 cells/ml. When an incompatible strain was used in the challenge inoculation, bacterial populations decreased more rapidly in the protected, symptomless tissues than in unprotected ones undergoing a hypersensitive reaction. These changes in bacterial populations indicate that protection may result from the release of inhibitory compounds into the intercellular spaces. In addition, cells of protected leaves appear to become more tolerant than normal cells to the presence of relatively high numbers of bacteria.