Detection of infection by Pseudomonas phaseolicola (Burkh.) Dowson in white-seeded dwarf bean seed stocks

Seeds of white-seeded varieties of dwarf beans infected with Pseudomonas phaseolicola may show fluorescent areas in the testa under ultra-violet light. Fluorescence is not visible in seed with dark-coloured testas and is masked by coloured seed dressings. Bacterial infection is not the only cause of fluorescence in testas and the type and colour of the fluorescence cannot be used to diagnose infection. Of 542 fluorescing seeds examined 112 were infected by the pathogen, and only six infected seeds were found in 495 non-fluorescing seeds from the same samples. It is estimated that in two seed stocks used for these experiments 64 and 68 % of infected seeds were of the fluorescent type. A 34 lb sample of seedstock has to be taken for examination to detect, with 95 % confidence, ten infected seeds in 1 cwt. The selection of all fluorescing seeds from such large samples effectively concentrates the infected seed into a sample small enough to be tested by bacteriological methods. Test seeds are examined bacteriologically by enrichment-culture in nutrient broth. Ps. phaseolicola is identified in these cultures from the diagnostic symptoms of halo-blight disease induced in bean seedlings inoculated with aliquots of the cultures.     

In vitro Production of Halo Blight Inducing Toxin by Pseudomonas phaseolicola (Burkh.) Dowson

Three pathogenic strains of Pseudomonas phaseolicola (strain 1 and 3 virulent and strain 5 weakly virulent) were tested for their toxic activity. All three strains produced detectable amounts of toxin in vitro. Cultural conditions and length of incubation greatly influenced toxin production. Maximum amount of toxin was produced at 20°C and pH 6.5. Glycerol served as the best carbon source and 1-cysteine as the best amino acid for toxin production.     

Production of the chlorosis inducing toxin in liquid cultures ofPseudomonas phaseolicola (Burkh.) Dowson

Amino acids affected amount and time course of the toxin production on complex as well as on chemically defined media. With glutamic acid the toxin content was high in the early growth stage but decreased later on, while the reverse was true with cysteine as single amino acid.Toxin production was highest at temperatures below 20°C. Generally, the toxin production started during the rapid growth phase and reached its maximum shortly after growth ceased. Chloramphenicolinhibited bacteria did not produce much toxin. Bacterial cells contained relatively low amounts of the toxin. Conditions for toxin production in large quantities are described. The feasibility to produce tritiummarked toxin was studied.Comparison of different strains showed that avirulent or weakly virulent halo-less strains do not produce detectable amounts of toxin. However, there was no correlation of the toxin production to the grouping of the bacterial strains in race 1 or the more virulent race 2.     

Multiple Hydrolases in Bean Leaves (Phaseolus vulgaris L.) and the Effect of the Halo Blight Disease Caused by Pseudomonas phaseolicola (Burkh.) Dowson

Multiple forms of hydrolytic enzymes were demonstrated in extracts of healthy bean leaves (Phascolus vulgaris L.) and bean leaves infected with the halo blight organism [Pseudomonas phaseolicola (Burkh.) Dowson] by polyacrylamide disc electrophoresis.Bean leaves contained up to 4 acid phosphatase bands, 9 esterase bands active towards alpha-naphthyl acetate, and 7 esterase bands towards alpha-naphthyl butyrate. Only low or no activity was found for alkaline phosphatase, lipase, and aminopeptidase.Two artifacts are described which were observed with the lead phosphate method for acid phosphatase and the Tween method for demonstration of lipase.After infection with the halo blight organism the major acid phosphatase of the host increased during early and decreased at later infection stages. An acid phosphatase of bacterial origin with a more neutral pH optimum could be demonstrated in infected leaves. It is suggested that the bacterial acid phosphatase plays a role in uptake of metabolites by the pathogen.Several esterase bands decreased after infection. One host band with activity towards alpha-naphthyl butyrate increased. Also the pathogen showed an esterase band with high activity towards alpha-naphthyl butyrate.     

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.     

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.     

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.     

Volatile Products of the Lipoxygenase Pathway Evolved from Phaseolus vulgaris (L.) Leaves Inoculated with Pseudomonas syringae pv phaseolicola

Activation of the "lipoxygenase pathway" in plants gives rise to a series of products derived from fatty acids. Analysis by gas chromatography-mass spectroscopy of volatile products produced by Phaseolus vulgaris (L.) cv Red Mexican leaves during a hypersensitive resistance response (HR) to the plant pathogenic bacterium Pseudomonas syringae pv phaseolicola showed evolution of several lipid-derived volatiles, including cis-3-hexenol and trans-2-hexenal, which arise from the 13-hydroperoxide of linolenic acid. These compounds were not produced in detectable amounts by buffer-inoculated leaves, nor did they evolve to such a high degree during comparable stages of the susceptible response. The absence of trans-2,cis-6-nonadienal, a product expected from 9-hydroperoxide of linolenic acid, suggests that lipid peroxidation during the HR proceeded primarily enzymically via bean lipoxygenase, which produces the 13-hydroperoxide, and not via autoxidative processes. The effects of trans-2-hexenal, cis-3-hexenol, and traumatic acid on P.s pv phaseolicola were investigaed. trans-2-Hexenal appeared to be highly bactericidal at low concentrations, whereas cis-3-hexenol was bactericidal only at much higher concentrations. Traumatic acid appeared to have no effect on P.s. pv. phaseolicola at the concentrations tested. These results demonstrate that during plant defense responses against microbial attack, several lipid-derived compounds are produced by the plant, some of which possess antimicrobial activity and conceivably are involved in plant disease resistance. The time of production of these substances, in amounts that would be expected to be antibacterial in vitro, correlated with a slowing down of the growth rate of bacteria in the leaves and was seen at a time before the accumulation of isoflavonoid phytoalexins in the host.     

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.     

Bioethanol production with carboxymethylcellulase of Pseudomonas poae using castor bean (Ricinus communis L.) cake

Increased consumption of fossil fuels is an emerging problem. Scientists look for the existence of other alternatives to fossil fuels, including so-called renewable energy. Accordingly, we report the production of bio-ethanol from the remnants of castor oil bean seed cake (CBC) by the carboxymethylcellulase enzyme (CMCase). A bacterial strain isolated from rice straw showing higher CMCase activity was identified. The 16S rRNA result showed a 93% homology with the 16SrRNA gene sequences of Pseudomonas poae RE11-1-14, the strain was identified as Pseudomonas poae AB3. In addition, our results showed that the highest enzyme activity was achieved after 48 h and inoculum size of 3.7 × 10⁵ CFU. The optimum temperature, pH and Carboxymethylcellulose (CMC) concentration for the highest enzyme activity was 25 °C, pH 7 and 10 g/l respectively. Furthermore, The CMCase was purified by ammonium sulphate at a concentration of 60%. The SDS-PAGE of the purified enzyme showed a molecular weight of 88 kDa. Additionally, the (CBC) was hydrolyzed by the purified CMCase at the enzyme optimum conditions. The results showed the liberation of 5.2 g/L of reducing sugar by using dinitrosalicylic acid (DNS) assay. Finally, the total sugar produces 35 g/L after 48 h when Saccharomyces cerevisiae was used as a fermentation agent. Hence for the first time, we have been successfully able to produce bioethanol from CBC with CMCase of Pseudomonas poae.     

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.     

Seed treatment for the control of halo-blight of beans (Pseudomonas phaseolicola)

Streptomycin and kasugamycin were applied as soaks, slurries or dusts to dwarf bean seeds either naturally (internally) infected or externally contaminated with Pseudomonas phaseolicola. Slurries of streptomycin (2–5 g a. i./kg seed) or kasugamycin (0–25 g a.i./kg seed) were the most effective treatments against both types of infection and were generally non-phytotoxic at these rates. Combined analysis of eight field experiments made over a 3 yr period showed that on average both compounds applied in slurries reduced primary infection from infected seeds by 98 %. The method thus shows considerable promise as a commercial control treatment.     

Inheritance of seed iron and zinc concentrations in common bean (Phaseolus vulgaris L.)

Micronutrients are essential elements needed in small amounts for adequate human nutrition and include the elements iron and zinc. Both of these minerals are essential to human well-being and an adequate supply of iron and zinc help to prevent iron deficiency anemia and zinc deficiency, two prevalent health concerns of the developing world. The objective of this study was to determine the inheritance of seed iron and zinc accumulation in a recombinant inbred line (RIL) population of common beans from a cross of low × high mineral genotypes (DOR364 × G19833) using a quantitative trait locus (QTL) mapping approach. The population was grown over two trial sites and two analytical methods (Inductively Coupled Plasma Spectrometry and Atomic Absorption Spectroscopy) were used to determine iron and zinc concentration in the seed harvested from these trials. The variability in seed mineral concentration among the lines was larger for iron (40.0–84.6 ppm) than for zinc (17.7–42.4 ppm) with significant correlations between trials, between methods and between minerals (up to r = 0.715). A total of 26 QTL were identified for the mineral × trial × method combinations of which half were for iron concentration and half for zinc concentration. Many of the QTL (11) for both iron (5) and zinc (6) clustered on the upper half of linkage group B11, explaining up to 47.9% of phenotypic variance, suggesting an important locus useful for marker assisted selection. Other QTL were identified on linkage groups B3, B6, B7, and B9 for zinc and B4, B6, B7, and B8 for iron. The relevance of these results for breeding common beans is discussed especially in light of crop improvement for micronutrient concentration as part of a biofortification program.     

The Translocation of 14C-Labelled Assimilates by Dwarf Bean Plants Infected with Pseudomonas phaseolicola (Burk), Dows

The distribution of ¹⁴C-labelled assimilate after infectionof the dwarf bean plant with Pseudomonas phaseolicola was followed.Infection of a single unifoliate leaf did not affect the totalfixation of ¹⁴CO₂ by unifoliates during the assimilation period.Fixation was maximal in unifoliates in the early stages of growthbut declined as trifoliates expanded. Unifoliates on infectedplants retained a greater proportion of assimilated ¹⁴carbonthan leaves on healthy plants.The pattern of distribution ofexported assimilate was not altered in the early stages of infection,the root and apex acting as the major sinks. As the diseasedeveloped, the first trifoliate leaf, unlike similar leaveson healthy plants, continued to import assimilate apparentlyat the expense of the root. Fixation by the first trifoliateand the distribution of assimilate from this leaf were not alteredby infection of a single unifoliate leaf. At no stage duringdevelopment of the disease was there any evidence of translocationof assimilate to either inoculated or non-inoculated unifoliates.