Growth of Pseudomonas phaseolicola in susceptible and in resistant bean plants

Race 1 of Pseudomonas phaseolicola introduced into leaves of susceptible Canadian Wonder bean plants multiplied logarithmically for 3–5 days, reaching final populations about 10⁵–10⁶ times the original. In resistant Red Mexican, Race 1 multiplied less rapidly to give final populations about 10²–10³ times the original. Race 2 behaved in susceptible Red Mexican as did Race 1 in Canadian Wonder. Macroscopic symptoms appeared in leaves when bacterial numbers reached their maxima. When introduced into the cotyledonary node Race 1 moved more rapidly upwards than downwards, and more rapidly and farther in Canadian Wonder than in Red Mexican. But even in Canadian Wonder the bacterium appeared only sporadically above the node of the first compound leaf. It could be isolated only rarely from chlorotic haloes around necrotic areas in leaves, or from chlorotic leaves not carrying lesions. Fewer lesions developed and the bacteria multiplied less in older than in younger leaves. Addition of glucose and casein hydrolysate to inocula of Race 1, separately or together, had little effect on growth in Canadian Wonder or Red Mexican, and the bacterium grew equally well in extracts of susceptible and of resistant plants. Preinoculation of leaves with an avirulent race reduced the number of lesions caused by a virulent race inoculated later, and also reduced growth of this race in leaves of a susceptible variety.     

Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease

The avrPphF gene was cloned from Pseudomonas syringae pathovar phaseolicola (PPH:) races 5 and 7, based on its ability to confer avirulence towards bean cultivars carrying the R1 gene for halo-blight resistance, such as Red Mexican. avrPphF comprised two open reading frames, which were both required for function, and was located on a 154 kb plasmid (pAV511) in PPH: Strain RW60 of PPH:, lacking pAV511, displayed a loss in virulence to a range of previously susceptible cultivars such as Tendergreen and Canadian Wonder. In Tendergreen virulence was restored to RW60 by avrPphF alone, whereas subcloned avrPphF in the absence of pAV511 greatly accelerated the hypersensitive resistance reaction caused by RW60 in Canadian Wonder. A second gene from pAV511, avrPphC, which controls avirulence to soybean, was found to block the activity of avrPphF in Canadian Wonder, but not in Red Mexican. avrPphF also conferred virulence in soybean. The multiple functions of avrPphF illustrate how effector proteins from plant pathogens have evolved to be recognized by R gene products and, therefore, be classified as encoded by avirulence genes.     

The relation between dosage, bacterial growth and time for disease response during infection of bean leaves by Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola 1L3 was infiltrated at a dosage of 0mD5 to 512 times the median effective dose (ED₅₀) into the leaves of two bean cultivars, Borlotto di Vigevano (ED₅₀ 15 bacteria) and Saluggia (ED₅₀ 34 bacteria). The distributions of time for production of disease symptoms after inoculation with up to 64 ED₅₀ were in agreement with those predicted by the simple birth-death model for microbial infection. Individual times of response to higher doses were longer and more widely distributed than expected from the model. Growth curves of bacteria in leaves inoculated with 16, 64 or 512 ED₅₀ could be viewed conventionally as a sequence of an exponential phase, a phase of decelerating growth and a stationary phase. Viable counts, however, were also compatible with parabolic population trends during the period preceding, accompanying and immediately following the appearance of disease symptoms. Bacterial growth parameters estimated from response times and infectivity titration data were consistent with those calculated from viable counts in vivo.     

Field studies on halo-blight of beans (Pseudomonas phaseolicola) and its control by foliar sprays

In 1968 when conditions were favourable for the spread of infection (rainfall 243 mm June-August), halo-blight of beans initiated from 0.38% primary infection produced 33.8% infected pods but in 1969 (rainfall 112 mm June -August+38 mm irrigation water), a similar level of primary infection resulted in only 3.2% infected pods. In both years plant and pod infection were reduced by approximately 90% by sprays of streptomycin sulphate or copper oxychloride (0.1% a.i. 60 gal/acre (675 l/ha)) applied every 10 days from seedling emergence to flowering time.     

Genetic and physiological variation among bean lines resistant and susceptible to bean anthracnose

Summary Electrophoresis was used to determine genetic and or biochemical variation, if any, among bean lines resistant and susceptible to anthracnose. This was based on two enzyme systems: peroxisase and esterase. It was revealed that resistant and suceptible plants differed in their band patterns and intensities. Band intensity differences occurred mainly among monomorphic bands with higher intensities expressed by susceptible plants, while band pattern differences were expressed both by resistant and susceptible plants. These differences appeared only at certain stages of development. These stages were identified as 3 and 40 days after emergence and were considered as critical stages for screening purposes. The peroxidase isozyme A₅ and the esterase isozyme C₁ at 3 days, and the peroxidase band C₁ and esterase bands A₁ and A₂ at 40 days were important because these differences could be used as genetic/biochemical markers for screening the population for resistance. Thus, electrophoretic differences could be used as a screening aid and this could save time and effort in breeding programmes. Comparisons between inoculated and non-inoculated leaves of resistant and susceptible lines indicated that infection induced changes in both the amount and kind of peroxidases even before symptoms of the disease appeared. However, there were no specific differences between resistant and susceptible lines, indicating that resistant and susceptible lines responded to infection in the same manner.     

Effectiveness of late copper and streptomycin sprays for the control of halo-blight of beans (Pseudomonas phaseolicola)

Two sprays of copper oxychloride or streptomycin sulphate (0–1 % a.i., 675 1/ha) applied to dwarf beans at first flowering and at pod set reduced pod infection by c. 70%. When applied as single sprays at pod set, copper was more effective than streptomycin and reduced pod infection by 50%. Although a copper spray at pod set increased the proportion of pods suitable for processing, more effective control was obtained with a regular spray programme, commencing at the seedling stage.     

Relationship between peroxidase activity and the amount of fully N-methylated compounds in bean plants infected by Pseudomonas savastanoi pv. phaseolicola

Changes in the level of endogenous formaldehyde (HCHO), some N-methylated compounds (choline and trigonelline) and peroxidase activity were examined in the leaves of bean genotypes (Phaseolus vulgaris L.) with different disease-sensitivity during ontogenesis in the stressfree condition and after natural infection by Pseudomonas savastanoi pv. phaseolicola (until the appearance of lesions). HCHO, as its dimedone adduct, and fully N-methylated compounds were determined by overpressured layer chromatography (OPLC) in different developmental stages and in the infected leaves/leaf discs. Peroxidase activity was measured by a spectrophotometric method. HCHO level decreased with ageing of the primary leaf and accordingly in the leaves at different developmental stages, then increased again in both cases due to the demethylation and methylation processes. Concentration of choline and trigonelline as potential HCHO generators decreased considerably while peroxidase activity increased with ageing of the plants. Comparing the symptomless and the Pseudomonas infected leaf discs (with watersoaked lesions) we found a decrease in the level of HCHO, choline and trigonelline and there was detectable increase in the peroxidase activity in the infected leaf tissues. Our findings are in accordance with previously published results that peroxidases play an important role in oxidative demethylation processes. Our hypothesis is that the high level of HCHO in the old leaves can originate from methylated components as the result of peroxidase activity and this high level may lead to the old leaf being resistant to pathogen. This conclusion is supported by the fact that the leaves of susceptible bean genotypes became resistant to Pseudomonas while growing older.     

Physiological and biochemical responses of resistant and susceptible wheat to injury by Russian wheat aphid

We examined the physiological and biochemical responses of resistant ('Halt' and 'Prairie Red') and susceptible ('TAM 107') wheat, Triticum aestivum L., to injury by the Russian wheat aphid, Diuraphis noxia (Mordvilko). Photosynthetic capacity was evaluated by measuring assimilation/internal CO2 (A/Ci) curves, chlorophyll fluorescence, chlorophyll, and nonstructural carbohydrate content. Total protein and peroxidase specific activity also were determined. No significant differences were detected in chlorophyll concentration between aphid-infested and control TAM 107 plants. The aphid-infested resistant cultivars had similar or significantly higher chlorophyll concentrations compared with their respective control plants. Measurements over time showed that infested Halt plants had delays in photosynthetic senescence, Prairie Red plants had photosynthetic rate changes that were similar to control plants, and TAM 107 plants displayed accelerated photosynthetic senescence patterns. The photochemical and nonphotochemical quenching coefficients were significantly higher in infested Halt plants compared with their respective control plants on day 3. Infested TAM 107 plants had significantly higher photochemical quenching compared with control plants at all times evaluated, and they had significantly higher nonphotochemical quenching on day 3. Throughout the experiment, infested Prairie Red plants exhibited photochemical and nonphotochemical quenching coefficient values that were not significantly different from control plants. Total protein content was not significantly different between aphid-infested and control plants for all cultivars. Differences between physiological responses of infested susceptible and resistant cultivars, particularly temporal changes in photosynthetic activity, imply that resistant Halt and Prairie Red wheat tolerate some impacts of aphid injury on photosynthetic integrity.     

A simple and efficient PCR method for the specific detection of Pseudomonas syringae pv phaseolicola in bean seeds

Using Southern blot hybridizations, it was found that the gene encoding the phaseolotoxin-insensitive ornithyl carbamoyl transferase (argK) was specific for Pseudomonas syringae pv. phaseolicola, the causal agent of the halo-blight disease. Based on these findings, a PCR protocol was developed for the specific detection of P.syringae. pv. phaseolicola in water-extracts of soaked bean seed. For this PCR protocol, two oligonucleotide primers were designed, based on the sequence of argK, which allowed the detection of a specific 1kb fragment. The protocol is simple since PCR was directly applied to bacterial suspensions, thus avoiding DNA extraction. The sensitivity of detection was increased by allowing the bacteria present in seed extracts to multiply in semi-selective media for 18h prior to PCR amplification. The detection threshold by visual detection using ethidium bromide staining was one naturally infected seed in lots of 400 to 600 seeds.     

Identification of quantitative trait loci involved in the response of common bean to Pseudomonas syringae pv. phaseolicola

Halo blight, caused by Pseudomonas syringae pv. phaseolicola (Burkn.) Downs (Psp), is an important disease in common bean (Phaseolus vulgaris L.). This study investigated the genetic control of the resistance to two local isolates of Psp (ITA-812 and ITA-684) in a recombinant inbred line (RIL) population derived from the cross between the bean genotypes Xana and Cornell 49242. The cultivar Cornell 49242 exhibited moderate resistance to these isolates, whereas cultivar Xana was susceptible. The RIL population showed a continuous variation in response to the two isolates. Analysis revealed four significant quantitative trait loci (QTLs): Psp4^812XC and Psp6.1^812XC located on linkage groups Pv04 and Pv06 (for the response to isolate ITA-812), and Psp6.1^684XC and Psp6.2^684XC located on Pv06 (for the response to isolate ITA-684). The QTLs Psp6.1^812XC and Psp6.1^684XC were located in the same genetic region (Psp6.1), close to the Psp6.2 region in which the QTL Psp6.2^684XC was mapped. A genetic dissection was undertaken to verify the consistency of these three QTLs located on the end of Pv06. Four sets of RILs were established according to the genotypes (Xana and Cornell 49242) of the underlying markers for the regions Psp6.1 and Psp6.2. Re-evaluation of these sets of lines revealed significant differences relative only to isolate ITA-684. The set of lines with the Cornell genotype in both regions was significantly more resistant than the other three sets of lines. This suggested that both regions were necessary to detect a significant effect in the response to isolate ITA-684. In the physical positions corresponding to these two genetic regions, in silico analysis revealed 16 candidate genes (putative orthologous genes) that carried sequences homologous to the resistance genes RPM1, FLS2, RPG1/RPG1-B, and Pto—all of which confer resistance to P. syringae in different species. The results confirm that, apart from the major genes implicated in resistance to Psp, specific bean genotypes exhibit a quantitative mode of inheritance of resistance to Psp.     

A qualitative difference in plasma renin activity in dahl rats susceptible or resistant to salt-induced hypertension

Plasma renin activity (PRA) was studied in the rats bred by Dahl for susceptibility (S-strain) or resistance (R-strain) to salt (NaCl) induced hypertension. The pH curves for PRA had different shapes. The difference in shape of the pH curves was reflected in the ratio of PRA pH 8/PRA pH 6.5. This ratio was shown to be characteristic of the strain and to be independent of changes in absolute PRA level induced by variation in dietary NaCl. The ratio of PRA pH 8/PRA pH 6.5 was also different between strains in weanling as well as adult rats. The underlying cause for the strain difference in the effect of pH on PRA is unknown, but may involve molecular differences between strains in either renin or renin substrate.     

Enzymes regulating the accumulation of active oxygen species during the hypersensitive reaction of bean to Pseudomonas syringae pv. phaseolicola

Changes in the activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC 1.11.1.7) and catalase (CAT; EC 1.11.1.6) which regulate the persistence of active oxygen species (AOS) were examined in leaves of bean (Phaseolus vulgaris L. cv. Tendergreen) undergoing compatible and incompatible interactions to race 6 and race 3 strains, respectively, of the halo-blight bacterium Pseudomonas syringae pv. phaseolicola. Resistance of cv. Tendergreen to race 3 is determined by the R3 gene and was expressed by a hypersensitive reaction (HR) which was associated with a rapid increase in lipid peroxidation between 8 and 12 h after inoculation. Five main isoforms of SOD were resolved by native polyacrylamidegel electrophoresis (PAGE). Major changes were found in the activities of the cytosolic Cu, Zn-SOD3 and Cu, ZnSOD5 isoforms, which increased by 6 h after inoculation with race 3, and the possibly peroxisomal MnSOD2 isoform, which decreased rapidly in tissue undergoing the HR. Three further minor isoforms of SOD showed a strong increase in activity during the HR. A low level of extracellular SOD activity was also resolved; two isoforms, one of which increased dramatically in activity during the HR, were detected within intercellular fluids recovered from inoculation sites. Fewer changes in SOD activities were found during the compatible interaction to race 6, and they did not occur until 16 h after inoculation. In tissue around infiltration sites, no decrease in the activity of Mn-SOD2 was observed but slight increases in some other isoforms were found. Four groups of POD isoforms were detected in both 3,3-diaminobenzidine/H₂O₂-and o-dianisidine/H₂O₂-stained PAGE gels. Significant changes in activity were again associated with development of the HR. In particular, by 2 h after inoculation, increases in POD3a, b and c isoforms were detected within total soluble extracts and also in POD3c within intercellular fluids (no other isoform was found in the apoplasm). By contrast, POD1 and POD2 activities generally declined following inoculation. The principal change in activity in tissues surrounding infiltration sites was an increase in POD3 isoforms following inoculation with race 3. Measurements of total activity showed a decrease in CAT activity as early as 2 h after inoculation, followed by a recovery after 8 h and a further decrease as infiltrated tissue collapsed during the HR. A more-gradual decline in CAT activity was observed at sites undergoing the compatible interaction and also in tissue surrounding inoculation sites. The spatial and temporal changes detected in activities of CAT and isoforms of SOD and POD clearly demonstrate the complexity and potential subtlety of control of the production and persistence of AOS in bean following microbial challenge. The generation of AOS through HR-specific, early increases in extra-cellular POD and SOD isoforms is discussed.This work was supported in part by the scientific Research Foundaation (OTKA F 5082), the foundation for Hungarian science, a british council scolership to A.L.A and the U.K. Agricultural and food Reaserch council.     

白纹伊蚊溴氰菊酯抗性和敏感品系羧酸酯酶性质比较

本文对白纹伊蚊Aedes albopictus溴氰菊酯抗性品系和敏感品系羧酸酯酶的生物化学性质进行了比较。白纹伊蚊抗性品系和敏感品系羧酸酯酶随底物浓度(α-乙酸萘酯或β-乙酸萘酯)的变化比活力变化趋势一致,但抗性品系对这2种底物的比活力均高于敏感品系,抗性品系羧酸酯酶的米氏常数和最大反应速度与敏感品系有显著差异。胆碱酯酶抑制剂测定结果表明,抗性品系羧酸酯酶对敌敌畏和磷酸三苯酯的敏感性高于敏感品系,对残杀威的敏感性低于敏感品系。2个品系羧酸酯酶对脱叶磷的敏感性差异不大。说明羧酸酯酶可能与白纹伊蚊对溴氰菊酯抗性有关。     

Multivesicular compartments proliferate in susceptible and resistant MLA12-barley leaves in response to infection by the biotrophic powdery mildew fungus

There is growing evidence that multivesicular bodies and cell wall-associated paramural bodies participate in the enhanced vesicle trafficking induced by pathogen attack. Here, we performed transmission electron microscopy in combination with cytochemical localization of H2O2 to investigate multivesicular compartments during establishment of compatible interaction in susceptible barley (Hordeum vulgare) and during hypersensitive response in resistant MLA12-barley infected by the barley powdery mildew fungus (Blumeria graminis f. sp. hordei). Multivesicular bodies, intravacuolar vesicle aggregates and paramural bodies proliferated in the penetrated epidermal cell during development of the fungal haustorium. These vesicular structures also proliferated at the periphery of intact cells, which were adjacent to the hypersensitive dying cells and deposited cell wall appositions associated with H2O2 accumulation. All plasmodesmata between intact cells and hypersensitive cells were constricted or blocked by cell wall appositions. These results suggest that multivesicular compartments participate in secretion of building blocks for cell wall appositions not only to arrest fungal penetration but also to contain hypersensitive cell death through blocking plasmodesmata. They may also participate in internalization of damaged membranes, deleterious materials, nutrients, elicitors and elicitor receptors.     

Blood biochemical factors in humans resistant and susceptible to formation of venous gas emboli during decompression

Blood biochemical parameters were measured in 12 male human subjects before and after exposure to a staged decompression protocol, with simulated extravehicular activity, during 3 days. Following the exposure, significant changes occurred in several parameters, including increases in blood urea nitrogen, inorganic phosphate, potassium, and osmolality, and decreases in uric acid and creatinine. Pre-exposure blood samples from subjects who were susceptible to formation of venous gas emboli (VGE) during decompression exhibited significantly greater levels of total cholesterol, high density lipoprotein cholesterol, potassium, inorganic phosphate, calcium, and magnesium. The results indicate that, following this decompression profile, small but significant (P less than 0.05) changes occur in several blood biochemical parameters, and that levels of certain blood factors may be related to susceptibility to VGE formation during decompression.     

Screening and biochemical characterisation of resistant and susceptible citrus species against dry root rot disease (Fusarium solani)

Fourteen citrus species were screened for their resistance against dry root rot under artificial inoculation conditions and classified as resistant (RHRL-122, RHRL-124, Australian sour, Sour dig, Balaji, Rangpurlime), moderately resistant (PKM-1, AL-Srirampur, Rough lemon), Susceptible (TAL 95/1, TAL 95/2, TAL 95/3, Nalgonda selection) and highly susceptible (TAL 94/13). The higher polyphenol oxidase (PPO) activity was observed in all infected leaves and roots of citrus species when compared to healthy leaves and roots at 15 and 30 days after inoculation (DAI). Higher PPO activity was observed in Balaji, Australian sour and Rangpurlime whereas lowest PPO activity was observed in TAL 94/13. In the case of leaf Peroxidase (PEO) isozyme profile an additional band which was darker and thicker was observed at an Em value of 0.24 in the case of Australia sour, Balaji and AL-Srirampur. In roots the PEO isozyme profile has the induction of single thick and darker additional band with an Em value of 0.47 was observed in Australian sour and Balaji. The banding profiles of estarase in leaves showed the induction of an extra band in the ase of Australian sour at Em 0.1, and at Em 0.53 in the case of Rangpurlime and Sourdig when compared to other species. The banding profile of esterase in roots was well expressed in Australian sour, PKM-1, Rough lemon, TAL-95/3, Rangpurlime and Sour dig. However, a minor band at Em 0.27 in Australian sour, TAL 95/1 and at Em 0.33 in Balaji was observed.