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.     

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.     

Epidemiology and strategy for the control of halo-blight of beans

A halo-blight epidemic model based on the equation of Van der Plank (1963) is described. The model has been used to determine tolerance levels for seed infection and to compare the effectiveness of chemical control measures based on foliar sprays and seed treatments.
Reduction of primary inoculum, derived from infected seed, by disease exclusion through seed testing, or by chemical seed treatment will give effective disease control under British conditions (i.e. with an expected apparent infection rate ( r ) of 0.15 per infected plant per day). Under climatic conditions more favourable for the disease where higher r values would occur, control treatments directed against primary inoculum only would be less effective. Under such conditions, it is predicted that chemical sprays, which have no effect on primary inoculum but cause a reduction in the rate of disease increase, would be more effective. The most effective disease control, however, would be a combination of treatments.     

A biochemical difference between healthy bean leaves resistant and susceptible to the halo-blight disease caused by Pseudomonas phaseolicola

Substrate for an endogenous oxidation in homogenates of leaves of bean (Phaseolus vulgaris) was traced to two fractions of lipids, each representing less than 2% of the dry weight of leaves. The substrate lipids, tentatively identified as galactosyl diglycerides, yielded linolenic and linoleic acids on acid hydrolysis. Amounts of linolenic acid in total lipids in resistant and susceptible leaves were similar. Amounts of free linolenic acid in resistant leaves increased eightfold to 408.6 μg and in susceptible leaves fourfold to 130.6 μg/g fresh leaf after homogenization and incubation for 16 min at 4 °C. These quantities are sufficient to account during their lipoxidation for the previously reported oxygen uptakes in homogenates. Differences between resistant and susceptible leaves were traced to the activities of lipase systems which liberate linolenic acid from substrate lipids.     

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.     

Sources and inheritance of resistance to halo-blight of Phaseolus beans

Forty-three accessions of Phaseolus vulgaris and four of P. coccineus were classified on the basis of the reaction of their primary leaves and pods to inoculation with races 1 and 2 of the pathogen Pseudomonas phaseolicola. Four groups were recognised, (1) highly resistant to race 1 only, (2) resistant to both races, (3) slightly resistant to both races or (4) susceptible to both races. Race 1 resistance was present in a number of accessions (e.g. Red Mexican UI3, Rona, Cornell 42–242). Resistance to race 1 and 2 was present in PI 150414, GN Nebraska No. 1 sel. 27, OSU 10183 and other accessions with PI 150414 in their parentage. Studies on the inheritance of resistance in Red Mexican UI3 and PI 150414 were made in crosses with various susceptible bean cultivars. A single dominant factor was responsible for race 1 resistance in Red Mexican UI3. The resistance factor in PI 150414 to races 1 and 2 behaved as a recessive in crosses with Cascade and Seafarer but was partially dominant in crosses with Gallatin 50. These findings suggest that genetic background can influence the expression of the resistance factor from PI 150414 and largely reconcile the conflicting evidence of previous investigators.     

Studies of halo-blight seed infection and disease transmission in dwarf beans

The transmission of Pseudomonas phaseolicola from plant to seed was mainly by the penetration of bacteria from external pod lesions to the underlying seeds. There was no evidence for translocation of bacteria from other parts of the plant to the seed, and symptomless pods contained only healthy seeds. Although a small proportion of infected seeds showed obvious symptoms of infection, the majority showed either slight symptoms, which could be detected only by careful observation, or were symptomless. In tests of disease transmission from seed to seedling, seeds with slight symptoms, or those which were symptomless were responsible for 35% and 52% respectively of the total disease transmission compared to 13% for obviously infected seeds. The viability of bacteria in seed stored under relatively uncontrolled conditions (10–27 C) declined by a factor of 250 per annum over the first 3 yr with extrapolation predicting effective extinction after c . 5 yr. The pathogen survived longer under controlled conditions (7–10 C and 45–50% r.h.) but no viable bacteria were detected in seed stocks which were 10-yr-old and with one exception 6-yr-old stocks were also free of the pathogen.     

Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola

Native EPS produced by Pseudomonas syringae pv. phaseolicola in vitro was separated by ion exchange chromatography on DEAE fractogel into three different polysaccharide fractions. A neutral polysaccharide eluting with the void volume yielded only fructose upon hydrolysis and exhibited an IR spectrum similar to authentic levan. At about 300 mM KCl a mannuronan eluted. Comparison with authentic alginate by IR spectroscopy, elution behaviour during DEAE-fractogel column chromatography, and monomer composition (mannuronic acid and traces of guluronic acid) confirmed the identity of this fraction as a bacterial alginate. It contained about 56 mol% acetyl groups. A third polysaccharide eluted at about 160 mM KCl. Its monomeric composition (rhamnose, fucose, glucose, and amino sugars), elution behaviour upon DEAE-fractogel column chromatography, and TLC patterns, closely resembled the sugar moiety of lipopolysaccharides (LPS) from, Pseudomonas syringae pv. phaseolicola. The protein component of crude EPS represented a fourth macromolecular fraction. It was not covalently linked to any of the polysaccharides since it could be removed from the EPS by phenol extraction.     

Bacteriophage and serological methods for the identification of Pseudomonas phaseolicola (Burkh.) Dowson

Rapid phage and serological methods were compared with biochemical and bean-pod inoculation tests for the identification of Pseudomonas phaseolicola. Phages II P, 12 P and 48 P were highly specific and did not lyse any of forty-five other isolates from bean or the sixteen species of Pseudomonas tested. They were also, however, unable to lyse the rough colony forms of P. phaseolicola which were occasionally isolated. Phage 12 S lysed a wide range of Pseudomonas spp., including a proportion of P. phaseolicola isolates, the majority of which were race 2. Serological tests showed that the heat-labile antigen possessed by P. phaseolicola was common to twelve other species of plant pathogenic pseudomonads. The heat-stable antigen was more specific and was detected only in P. mors-prunorum and P. primulae, neither of which occurs on bean. The two races of P. phaseolicola were indistinguishable in serological tests. It was concluded that both phage and antiserum tests provide specific, rapid and easily applied methods for the routine identification of P. phaseolicola isolates from plant and seed material.     

Resistance to halo-blight in the Cambridge accessions V4604 and V4058 of Phaseolus beans

V4604, a Cambridge University accession of Phaseolus vulgaris, had a high degree of leaf resistance to race 1 of Pseudomonas phaseolicola and partial resistance to race 2. Genetic control of resistance to race 1 was by a major dominant gene which is also present in the cv. Red Mexican. The partial resistance of V4604 to race 2 was under polygenic control, as was the low level resistance to race 2 in V4058.     

Growth Responses of Dwarf Bean to Infection by Pseudomonas phaseolicola (Burk) Dows

At an early growth stage inoculation of both unifoliates ofdwarf beans with Pseudomonas phaseolicola (Burk) Dows. Race1 inhibited trifoliate production, but inoculation of one unifoliateonly reduced subsequent leaf expansion, and the opposite uninfectedunifoliate became significantly larger than unifoliates on uninfectedplants. Inoculation of the first or second trifoliates or thecotyledonary node at a later developmental stage all reducedprimary-shoot growth by 25 to 30 per cent, but there were markeddifferences in the extent of secondary-shoot development inleaf axils below the inoculation point. The growth effects were related to the distribution and activityof the bacterial toxin within the plant, particularly to differencesin the rate of invasion of apical regions. The primary toxineffects appear to be on apical function and leaf expansion.     

Molecular analysis of thermoregulation of phaseolotoxin-resistant ornithine carbamoyltransferase (argK) from Pseudomonas syringae pv. phaseolicola

The phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) and phaseolotoxin are produced by Pseudomonas syringae pv. phaseolicola at 18 degrees C but not at 28 degrees C. At 28 degrees C, the pathogen produces a protein(s) that binds (in vitro) to a 485-bp fragment (thermoregulatory region, TRR) from a heterologous clone from the pathogen genomic library, which in multiple copies overrides thermoregulation of phaseolotoxin production in wild-type cells (K. B. Rowley, D. E. Clements, M. Mandel, T. Humphreys, and S. S. Patil, Mol. Microbiol. 8:625-635, 1993). We report here that DNase I protection analysis of the 485-bp fragment shows that a single site is protected from cleavage by the protein in the 28 degrees C extract and that this site contains two repeats of a core motif G/C AAAG separated by a 5-bp spacer. Partially purified binding protein forms specific complexes with a synthetic oligonucleotide containing four tandem repeats of this motif. A 492-bp upstream fragment from argK encoding ROCT also forms specific complexes with the protein in the 28 degrees C crude extract, and a 260-bp subfragment from the TRR containing the binding site cross competes with the argk fragment, indicating that the same protein binds to nucleotides in both fragments. DNase I protection analysis of the fragment from argK revealed four separate protected sequence elements, with element III containing half of the core motif sequence (CTTTG), and the other elements containing similar sequences. Gel shift assays were done with DNA fragments from which one or all of the sites were removed as competitor DNAs against the argK probe. The results of these experiments confirmed that the binding sites (in argK) are necessary for the protein to bind to the argK fragment in a specific manner. Taken together, the results of studies presented here suggest that in cells of P. syringae pv. phaseolicola grown at high temperature argK may be negatively regulated by the protein produced at this temperature.     

Genetic transformation of Pseudomonas phaseolicola by R-factor DNA.

Summary The bacterium Pseudomonas phaseolicola was successfully transformed, for the first time, with R-factors RSF1010 and pBR322 DNA by a calciumshock and heat-pulse technique. Frequency of transformation for RSF1010 ranged from 0.8×10^-7 to 3.1×10^-6 and was ca. 0.4×10^-8 for pBR322.     

Plant and environmental sensory signals control the expression of hrp genes in Pseudomonas syringae pv. phaseolicola.

The hrp genes of Pseudomonas syringae pv. phaseolicola control the development of primary disease symptoms in bean plants and the elicitation of the hypersensitive response in resistant plants. We examined the expression of the seven operons located in the 22-kb hrp cluster (L. G. Rahme, M. N. Mindrinos, and N. J. Panopoulos, J. Bacteriol. 173:575-586, 1991) in planta and in vitro under different physiological and nutritional conditions by using chromosomally located hrp::inaZ reporter fusions. We show that (i) a plant signal(s) is specifically required for the induction of the seven hrp operons, during both compatible and incompatible interactions; (ii) hrpL and hrpRS are regulated by different mechanisms in planta and in vitro; and (iii) expression of individual hrp loci is differentially affected by pH, osmotic strength, and type of carbon source: hrpAB, hrpC, and hrpD were downregulated similarly by osmolarity, pH, and certain carbon sources; hrpE expression was affected strongly by pH and carbon substrate and slightly by osmolarity; and hrpF was not substantially affected by any of these factors. These findings suggest complex signaling mechanisms taking place during plant-pathogen interactions.     

Variation in the Virulence of some Streptomycin Resistant Mutants of Pseudomonas phaseolicola

One strain of each of the 2 races of Pseudomonas phaseolicola was tested for in vitro resistance to streptomycin. The race 1 strain showed a greater resistance to the antibiotic than the race 2 strain. Resistant mutants were isolated from each strain and used to infect plants of Phaseolus vulgaris cv. Higuerillo V 345. Variation was noted in both the range of symptoms produced and in the numbers of bacteria re-isolated from the plants. Some mutants retained their resistance after passage through the plant while others reverted back to parental type.