Halo Blight Research Articles

Pathogenic Variation in Halo Blight on Common Beans

Halo blight, caused by a bacterium, Pseudomonas syringae pv. phaseolicola (Psp), is one of the most important bacterial diseases of common beans (Phaseolus vulgaris L.). The objective of this study was to evaluate the pathogenic variation of Psp strains with emphasis on those collected in Nebraska. Twenty-nine Psp strains were inoculated on 20-day-old leaves of eight differential cultivars/lines and a resistant check line great northern (GN) Nebraska #1 sel. 27. Two experiments were conducted in growth chambers under 20 °C and 12-h photoperiod. A split-plot design was used with nine cultivars/lines as whole plots in a randomized complete block design (RCBD) and strains as sub-plots in an incomplete block arrangement. Data were analyzed as a split-plot with a RCB arrangement because incomplete block was not significant. Strains were classified into five races with six, 16, and two strains classified as races 1, 6, and 7, respectively. The remaining strains were designated tentatively as new races 10 and 11. GN Nebraska #1 sel. 27 was resistant to all Psp strains tested. This information is useful to breeders interested in developing germplasm and cultivars resistant to Psp.

Characterization of Halo Blight Races on Dry Beans in South Africa.

Isolates of the halo blight pathogen Pseudomonas syringae pv. phaseolicola were collected in the bean-producing areas in South Africa from 1991 to 1996. Of the 1,128 isolates collected, 967 were identified as P. syringae pv. phaseolicola. The majority of these isolates were obtained from a wide range of Phaseolus vulgaris cultivars, and the rest from P. coccineus and P. lunatus. Two hundred fifty-five isolates, representative of all the localities and cultivars sampled, were categorized into different races according to their reaction on a set of differential cultivars. Seven races (1, 2, 4, 6, 7, 8, and 9) were identified, with race 8 the most prevalent. Races 1, 2, 6, and 8 were widely distributed through the whole production area, while races 4, 7, and 9 were restricted to one or two localities.

Construction of a Genetic Linkage Map and Locations of Halo Blight and Brown Spot Resistance Loci in Common Bean (Phaseolus vulgaris L.) using RAPD Markers

Halo blight (HB), brown spot (BS), and rust incited by the bacterial pathogens Pseudomonas syringae pv. phaseolicola (Psp), Pseudomonas syringae pv. syringae (Pss) and the fungal pathogen Uromyces appendiculatus, respectively, are important diseases of common beans. The objectives were to construct a RAPD linkage map, and to locate HB and BS resistance genes and genes for some other traits. One-hundred-seventy RAPD markers were mapped in 78 RI lines of the cross BelNeb 1 and A 55. Eleven main and nine minor linkage groups were identified. MAPMAKER/QTL, interval mapping, was used to identify genomic regions involved in the genetic control of the traits. One region was found to control HB leaf reactions to strain HB16 while three regions controlled reactions to strain HB 83. These regions accounted for 22% and 18%, 17%, and 17% of phenotypic variation of resistance, respectively. Four putative QTLs were identified for resistance to BS, and accounted for 37%, 26%, 23%, and 19% of the phenotypic variation. Rust resistance was determined by a single major gene to both rust strains US85NP 5-1 and D82vc74fh. However, linked markers were not identified. The V gene controlling flower and stem color was tightly linked with the Operon marker O10.620.

Breeding Great Northern and Pinto Dry Beans with Multiple Disease Resistance Combined with Improved Seed Quality, Adaptation, Yield, and Plant Type

Common bacterial blight (CBB), rust (RU), and white mold (WM) are serious diseases of great northern (GN) and pinto (P) beans in Nebraska and Colorado. The bacterial diseases halo blight (HB) and brown spot (BS) are sporadic. Severe Fe-induced leaf chlorosis (Fe ILC) occurs on calcareous sites. Separate inoculated disease nurseries are used to screen for resistance to the pathogens causing the above diseases. Yields and seed quality of lines are also determined in non-disease trials. Sources of exotic resistance to the above pathogens and to Fe ILD have been identified and their inheritance determined. A non-structured recurrent selection scheme has mainly been used, occasionally with a backcross program, to combine high levels of the desired traits. Selection for highly heritable traits such as seed size, shape and color, maturity, plant architecture, and RU resistance occurs in early generations while traits of low heritability, such as CBB resistance, WM avoidance, yield, seed coat cracking resistance, and canning quality, are evaluated in separate replicated tests over several years and finally for yield in on-farm-trials. A number of multiple disease resistant, high-yielding, well-adapted GN and P lines are or will be released; P `Chase' (on about 30,000 acres in 1996) and GN WM 3-94-9 (for possible release).

Inheritance of Resistance to Halo Blight Flower and Stem Color and Association in Common Beans

Halo blight is one of the most important bacterial diseases of common beans (Phaseolus vulgaris L.). It is serious under moderate temperature and high humidity conditions. The disease is caused by a seed-borne bacterium, Pseudomonas syringae pv. phaseolicola (Burkh.) Dowson (Psp). The inheritance of leaf reactions to Psp, flower, and stem color was studied using greenhouse-grown 109 F9 recombinant inbred lines (RI) from the P. vulgaris cross BelNeb 1 [resistant (R)] (USDA/NE) × A 55 [susceptible (S)] (CIAT). Two Psp strains, HB16 (NE) and 83-Sc2A (NE), were inoculated using the water-soaking method. A segregation ratio of 1 R:1 S RI lines were observed for disease reactions in leaves for both strains indicating major gene control. The presence of recombinants for SR, RS to the strains indicated that different genes were involved. Stem (SC) and flower (FC) color traits were each determined by two major genes. Linkages were found for reactions to the two Psp strains and also between FC and SC. No linkages were observed from FC and also SC with reactions to Psp strains.

A Rapid and Sensitive PCR-Based Assay for Concurrent Detection of Bacteria Causing Common and Halo Blights in Bean Seed

A quick polymerase chain reaction (PCR)-based procedure was developed for concurrent detection of the causal agents of bean common blight, Xanthomonas campestris pv. phaseoli, and bean halo blight, Pseudomonas syringae pv. phaseolicola, in bean seed. A rapid DNA extraction procedure, consisting of briefly wetting intact or crushed seeds with a solution of sodium hydroxide, was used to extract bacterial DNA from externally and internally blight-contaminated seeds. G+C-rich oligonucleotide primers were designed from the phaseolotoxin gene cluster of P syringae pv. phaseolicola and tested under high-stringency conditions in PCR assays. The HB14 primers specifically directed the amplification of a 1.4-kb fragment from DNA of 19 P syringae pv. phaseolicola isolates, whereas templates from 62 other bacterial strains, including the bean pathogens P syringae pv. syringae and X. campestris pv. phaseoli, and plant-pathogenic species of Agrobacterium, Clavibacter, Erwinia, and Xanthomonas did not produce any discrete bands upon amplification. The HB14 primers for P. syringae pv. phaseolicola, and the previously reported G+C-rich X4 primers, specific for X. campestris pv. phaseoli, amplified discrete DNA fragments by soaking extracts of white bean seeds contaminated with halo and common blights, respectively. DNA extracts from blight-infested colored seeds were recalcitrant to PCR-amplification unless polyvinylpyrrolidone was added to the extraction buffer. In combination, X4 and HB14 primers successfully detected individual and mixed infections of bean common and halo blights and yielded distinctive DNA fragments from batches containing as few as 1 infected seed in 10,000 seeds.

Effectiveness of systemic resistance in bean against foliar and soilborne pathogens as induced by biological and chemical means

Unifoliate leaves of 9‐day‐old green bean, Phaseolus vulgaris cv. Redlands Pioneer, were inoculated with 104 conidia/ml Colletotrichum lindemuthianum, causing local lesions, or sprayed with 20 μg 2, 6‐dichloro‐isonicotinic acid/ml formulated by Ciba‐Geigy Ltd as CGA 41396. At various times afterwards (7–16 days), first, second or third trifoliate leaves of these plants were challenge‐inoculated with 105 conidia/ml C. lindemuthianum or with the rust pathogen, Uromyces appendiculatus. The numbers of anthracnose lesions or rust uredinia resulting from challenge‐inoculation were reduced to similar extents by both pre‐treatments compared with control plants. Halo blight, caused by Pseudomonas syringae pv. phaseolicola, was reduced in first trifoliates following treatment of unifoliate leaves 6 days earlier with CGA 41396. Induced resistance to root‐infecting pathogens was not observed when stems of either 14‐ or 16‐day‐old plants were inoculated with mycelial plugs of Fusarium solani f. sp. phaseoli, or when 11‐ and 15‐day‐old plants were inoculated with Rhizoctonia sp., Treatment with CGA 41396 did not protect seedlings when they were transplanted into a mix containing the Fusarium sp. 1 day later.

Genetic organization of a cluster of genes involved in the production of phaseolotoxin, a toxin produced by Pseudomonas syringae pv. phaseolicola

Phaseolotoxin [N delta(N'-sulfo-diaminophosphinyl)-ornithyl-alanyl- homoarginine] produced by Pseudomonas syringae pv. phaseolicola, the bean halo blight pathogen, is a potent inhibitor of ornithine carbamoyltransferase (OCT). Inhibition of OCT in infected plants leads to chlorosis and growth inhibition. A genomic cosmid clone, pHK120, containing a 25-kb fragment of DNA from a wild-type strain of P. syringae pv. phaseolicola restores toxin production in Tox- mutants. Tn5 mutagenesis of pHK120 and marker exchange of pHK120::Tn5 plasmids in the wild-type strain resulted in the isolation of 39 chromosomal mutants that harbor Tn5 insertions at known positions. Toxin bioassays revealed that 28 of the mutants, with Tn5 insertions distributed throughout the insert of pHK120, were Tox-, indicating that a functional locus for toxin production in each mutant was inactivated. Complementation analysis was done by testing for toxin production strains that carried a genomic Tn5 at one location and a plasmid-borne Tn5 at another location (pair complementation). Pair complementation analysis of nine marker exchange mutants and a random genomic Tn5 mutant revealed that there are a minimum of eight toxin loci (phtA through phtH) in pHK120. Mutants carrying Tn5 insertions in the phtA, phtD, and phtF loci were complemented by deletion subclones containing fragments from pHK120; mutants carrying Tn5 insertions in the phtC locus were partially complemented by a subclone, and mutants carrying Tn5 insertions in the phtB, phtE, phtG, and phtH loci were not complemented by any of the available subclones. A comparison of the insert from pHK120 with that from pRCP17, a clone reported previously (R. C. Peet, P. B. Lindgren, D. K. Wills, and N. J. Panopoulos, J. Bacteriol. 166:1096-1105, 1986) by another laboratory to contain some of the phaseolotoxin genes and the phaseolotoxin-resistant OCT gene, revealed that the inserts in these two cosmids overlap but differ in important respects.

The cloning and characterization of phage promoters, directing high expression of luciferase in Pseudomonas syringae pv. phaseolicola, allowing single cell and microcolony detection

Regions of DNA containing promoter sequences from a Pseudomonas syringae pv. phaseolicola-specific phage (phi 11P) were identified by shotgun cloning into a broad-host-range promoter-probe vector (pQF70). When used in conjunction with the luciferase reporter genes, one of these DNA fragments, 19H, directed gene expression at a level which enabled the subsequent light output (bioluminescence) of single cells of P. syringae pv. phaseolicola to be detected and visualized using a charge-coupled device (CCD). The P. syringae pv. phaseolicola phi 11P, 19H and P. aeruginosa phi PLS27, HcM promoters gave a 50-fold increase in bioluminescence (maximum relative light output) compared to similar constructs containing other well-characterized promoters, for example, tetracycline. Similar bioluminescent characteristics of the transformed bacterium, were observed during growth with and without antibiotic-selection. When lux+ bacteria were inoculated onto French bean leaf (Phaseolus vulgaris L.), the resultant secondary halo blight lesions were bioluminescent and during phylloplane colonization by the lux+ bacterium, bioluminescence on leaf surfaces was detected and imaged by the CCD. Use of these newly identified promoters, combined with the greatly increased sensitivity of bioluminescence detection by the CCD, thus provided a new dimension for the study of natural ecological populations during the bacterial colonization of plants.

Generalized Induction of Defense Responses in Bean Is Not Correlated with the Induction of the Hypersensitive Reaction.

Transcripts for phenylalanine ammonia-lyase, chalcone synthase, chalcone isomerase, and chitinase accumulated in common bean after infiltration with the Pseudomonas syringae pv tabaci Hrp- mutant Pt11528::Hrp1, even though a hypersensitive reaction did not occur. The temporal pattern of this transcript accumulation was similar to that seen after infiltration with wild-type P. s. tabaci Pt11528, which resulted in a hypersensitive reaction. Escherichia coli DH5[alpha], P. fluorescens Pf101, heat-killed Pt11528 cells, and Pt11528 cells treated with protein synthesis inhibitors also induced accumulation of defense transcripts but not a hypersensitive reaction. In contrast, these transcripts were not detected in plants infiltrated with water or P.s. pv phaseolicola NPS3121, a compatible pathogen that causes halo blight. Phytoalexins were produced in bean after infiltration with Pt11528, Pt11528::Hrp1, Pt11528 cells treated with neomycin, or Pf101, but not in plants infiltrated with NPS3121 or water. These results suggest that there are unique biochemical events associated with the expression of a hypersensitive reaction which are distinct from other plant defense responses such as phytoalexin biosynthesis. In addition, our results support the hypothesis that there is a general, nonspecific mechanism for the induction of defense transcripts and phytoalexins by pathogenic and saprophytic bacteria that is distinct from the more specific mechanism associated with the induction of the hypersensitive reaction.

Specific Detection ofPseudomonas syringaepv.phaseolicolaDNA in Bean Seed by Polymerase Chain Reaction-Based Amplification of a Phaseolotoxin Gene Region

The polymerase chain reaction (PCR) is described for the specific detection of the bean halo blight pathogen Pseudomonas syringae pv. phaseolicola in bean seed. The method involves the amplification of a segment of the tox (phaseolotoxin) gene cluster (23). Two oligonucleotide primers, designed according to the sequence of this segment, allowed the exclusive amplification of a 1.9-kb fragment from total DNA extracted from pure cultures of the pathogen and from water extracts of soaked bean seed. The amplified segment was visually detected by ethidium bromide staining when the original samples contained approximately 10 3 colony-forming units (cfu) per milliliter and by Southern blot hybridization in DNA extracts from pure cultures of the pathogen that contained as few as 10 cfu/ml [...]

Antibacterial Activity of Tea and Coffee Wastes Against Some Plant Pathogenic Pseudomonas syringae Strains

In vitro and greenhouse trials were conducted to elucidate the potential use of extracts of tea and coffee wastes to control plant diseases caused by the bacterial pathogens, Pseudomonas syringae pv. pisi (race 1 and 2) and P. s. pv. phaseolicola (race 1). The antibacterial activity was measured as the diameter of the inhibition zone in agar and also by periodical viable cell counts in laboratory tests. The effect on the hypersensitive reaction and the potential for disease control after leaf infiltration and seed treatment were studied on bean plants in the greenhouse. Results showed that both the tea and coffee extracts possessed antibacterial activity against the three pathogens, but that the effects varied depending on the strain and the test method. Strong reduction of halo blight disease and improvement in plant growth was obtained in presence of the coffee extract. For the halo blight pathogen, P. s. pv. phaseolicola, there was a good correlation between the results from the viable cell count method and the greenhouse tests, but the results from the in vitro studies did not agree with those from greenhouse as regards the P. s. pv. pisi strains. It is suggested that the component(s) in tea and coffee responsible for controlling the bean pathogen may not be the same as that for the pea pathogens.

Promotion of bacterial infection of leaves by an organosilicone surfactant: Implications for biological weed control

Abstract A nonionic organosilicone surfactant (Silwet L-77) capable of facilitating stomatal penetration by aqueous solutions was evaluated for its capacity to enhance bacterial infections. Disease levels and bacterial populations were evaluated with the halo blight pathogen Pseudomonas syringae pv. phaseolicola (P.s.p.) on bean (Phaseolus vulgaris) using different rates of surfactant (0–0.25% v/v) and pathogen numbers (log 5–8 cfu/ml). Maximum levels of disease severity and pathogen populations in leaf tissue were obtained with a surfactant rate of ⩾0.2% and log 8 P.s.p. High levels of disease were achieved under hot, dry conditions (29°C, 55% RH) at the time of pathogen application. No disease was observed when the pathogen was applied without surfactant, regardless of pathogen numbers. An optimal rate of surfactant (0.2%) plus pathogen (log 8) was then tested on the weed kudzu (Pueraria lobata) and shown to enhance disease. Hydathode infection of cabbage (Brassica oleracea) by Xanthomonas campestris pv. campestris (X.c.c.) was studied with and without surfactant, utilizing a bioluminescent strain (FD911) for visualization of the colonization patterns of the leaf. Hydathode infections were visible as distinct bright spots on the leaf margin, while stomatal infections were visible as points of light scattered across the lamina. This model system provides data which indicate that bacterial infection and disease severity can be enhanced by using a stomata-penetrating surfactant and that this technology has potential applications in biological control.

Pathovar-specific requirement for the Pseudomonas syringae lemA gene in disease lesion formation.

The lemA gene is conserved among strains and pathovars of Pseudomonas syringae. In P. syringae pv. syringae B728a, a causal agent of bacterial brown spot disese of bean, the lemA gene is required for lesion formation on leaves and pods. Using lemA-containing DNA as a probe, we determined that 80 P. syringae pv. syringae strains isolated from bean leaves could be grouped into seven classes based on restriction fragment length polymorphism. Marker exchange mutagenesis showed that the lemA gene was required for lesion formation by representative strains from each restriction fragment length polymorphism class. Hybridization to the lemA locus was detected within six different P. syringae pathovars and within Pseudomonas aeruginosa. Interestingly, a lemA homolog was present and functional within the nonpathogenic strain P. syringae Cit7. We cloned a lemA homolog from a genomic library of P. syringae pv. phaseolicola NPS3121, a causal agent of halo blight of bean, that restored lesion formation to a P. syringae pv. syringae lemA mutant. However, a lemA mutant P. syringae pv. phaseolicola strain retained the ability to produce halo blight disease symptoms on bean plants. Therefore, the lemA gene played an essential role in disease lesion formation by P. syringae pv. syringae isolates, but was not required for pathogenicity of a P. syringae pv. phaseolicola strain.

Races and Survival ofPseudomonas syringaepv.phaseolicolain Northern Tanzania

The pathogenic variation among 118 isolates of Pseudomonas syringae pv. phaseolicola associated with halo blight of bean (Phaseolus vulgaris) in the Arusha and Kilimanjaro regions of northern Tanzania from November 1988 to February 1990 was examined with four differential bean cultivars. The three races recovered were race 2 (52.5%), race 1 (45.0%), and race 3 (2.5%) Some strains of race 2, designated as 2P, produced a brown, diffusible pigment on agar media. Some race 2P strains grew at 34 C, whereas the non-pigment-producing strains of race 2 and of races 1 and 3 failed to grow at this temperature (.)

Pseudomonas syringaepv.phaseolicolaPopulations and Halo Blight Severity in Beans Grown Alone or Intercropped with Maize in Northern Tanzania

In tests in northern Tanzania, populations of Pseudomonas syringae pv. phaseolicola on and in bean (Phaseolus vulgaris) foliage were higher, severity of halo blight on foliage and pods was greater, and incidence of seed infection was higher when beans were intercropped with maize (Zea mays) than when beans were grown alone. Maize leaves did not support high populations of the pathogen and thus did not seem to provide additional inoculum for the bean crop when the two were grown in association. However, bean leaves in the intercrop system took longer to dry after rainfall and were wetter than those in the monoculture (.)

The Role of Weeds in Survival ofPseudomonas syringaepv.phaseolicolain Northern Tanzania

Of 16 weed species belonging to 10 families, only Neonotonia wightii served as a perennial reservoir of Pseudomonas syringae pv. phaseolicola, the cause of halo blight. The weed survived dry periods along fences and hedgerows, by roadsides, on ditch banks, and in corners of farmers' fields. Only race 1 of P. s. phaseolicola was recovered from N. wightii. With artificial inoculation, however, all three races of the bacterium caused lesions in the weed. Because race 1 of P. s. phaseolicola was not seedborne in N. wightii, the weed cannot spread the pathogen into new areas by means of seed dispersal

Occurrence of Bacterial Halo Blight of Rice in Hokkaido, Japan

Occurrence of Bacterial Halo Blight of Rice in Hokkaido, Japan

Non‐pathogenic association of L‐form bacteria(pseudomonas syringae pv. phaseolicola)with bean plants(Phaseolus vulgarisL.) and its potential for biocontrol of halo blight disease

L‐forms of the halo blight pathogen, Pseudomonas syringae phaseolicola, were maintained in a medium which suppressed cell wall synthesis. These L‐forms, unlike revertants (walled forms derived from unstable L‐forms) and cell walled (parent) organisms, did not elicit a hypersensitive response in tobacco leaves. Association of L‐forms with Phaseolus vulgaris was established by seed imbibition in L‐form suspensions compared with appropriate control treatments (5% mannitol or heat‐killed cells). Seedling emergence and plant growth was not affected by L‐form imbibition. The association was detected by agglutination assays using polyclonal antibody. The L‐form association was localized to the lower shoot tissue and was progressively lost with age of plants. Plants with associated L‐forms had vigour and shoot weights equivalent to controls and showed no disease symptoms. The cell walled form could not be isolated from plants showing positive agglutination. On challenge with the pathogen, plants associated with L‐forms showed significantly less disease symptoms than controls. Stem extracts, from associated plants, were inhibitory to in vitro cultures of both L‐forms and parent forms of Ps. syr. phaseolicola. These results indicate that L‐form associations confer induced systemic resistance to bean plants and might be developed as novel biocontrol systems.

A rapid screening technique to combine resistance to halo blight and bean common mosaic virus in Phaseolus vulgaris L.

Seven bean lines (Phaseolus vulgaris L.) with differential resistance or susceptibility to race 2 of halo blight (Pseudomonas syringae pv. phaseolicola) and a necrosis-inducing isolate of bean common mosaic virus were inoculated with one or both pathogens in combination, to determine the feasibility of dual screening to identify resistance to both pathogens simultaneously. Dual screening yielded the same results as separate screenings. Neither pathogen affected the disease expression of the other. Simultaneously screening for resistance to both pathogens will shorten the recurrent screening-selection cycle of hybridization programs, and accelerate development of resistant cultivars.