Genetics of specific resistance in pea (Pisum sativum) cultivars to seven races of Pseudomonas syringae pv. pisi

Seven races of Pseudomonas syringae pv. pisi were distinguished using eight differential cultivars of pea (Pisum sativum). Segregation among F₂ populations of crosses between differential cultivars sequentially inoculated with races of P.s. pv. pisi provided evidence for four and possibly six putative resistance(R)/avirulence(A) gene pairs. R1, R2 and R3 are dominant resistance alleles at single loci, R4 is a dominant allele at a single locus which exhibits variable expression possibly dependent on genetic background. There is evidence that R3 and R4 are at linked loci. Homology tests provided proof of the occurrence of the alleles R2, R3 and R4 in more than one cultivar. Two other alleles, R5 and R6, were postulated to explain the observed segregation ratios in certain crosses.
It can be inferred that P.s. pv. pisi races 2, 3 and 4 each carry a different single a virulence gene, race 6 carries no apparent avirulence genes, and race 7 carries at least A2, A3 and A4. Race 1 carries Al, A3, A4 and possibly A6; race 5 carries A2, A4 and possibly A5 and A6.     

Evaluation of physiological and serological profiles of Pseudomonas syringae pv. pisi for pea blight identification

Phenotypic variability of the pea blight bacterium, Pseudomonas syringae pv. pisi, was studied on a large collection of strains isolated in France, as well as those obtained from foreign collections. Some other pseudomonads encountered on peas, particularly P.s. pv. syringae, were included in the study to evaluate differential tests for identification purposes. All the isolates that induced watersoaking on the pea cultivar Kelvedon Wonder after inoculation were considered to be P.s. pv. pisi. The other pseudomonads gave either no reaction or a hypersensitive reaction. When they corresponded to P. syringae according to the LOPAT test, they were referred to as P.s. pv. syringae.
P.s. pv. pisi did not show a single uniform phenotype. The variation of the different tests was estimated (fluorescence+ 93%; esculin-86%; dl-lactate-85%; homoserine + 75%; INA + 97%). Three O-sero-groups contained P.s. pv. pisi strains: APT-PIS (88.5%), HEL2 (11.4%) and RIB (0.1%). When the main criteria were combined, eight profiles were encountered within P.s. pv. pisi. This diversity was not linked to race structure or geographical origin of the strains. Profile PI was the most frequent (72.8%), and it was specific to the pathovar pisi . The strains belonging to the other profiles could be confused with some P.s. pv. syringae strains because of the serological heterogeneity of that pathovar. For instance, the pv. pisi strains belonging to profiles P2 and P4 resembled some of the P.s. pv. syringae found on peas and required pathogenicity tests on pea for their identification. The confusing pea isolates represented 12.8% of the total 4740 strains studied.     

Distinguishing pathovars of Pseudomonas syringae on peas: nutritional, pathogenicity and serological tests

Of the published methods to distinguish Pseudomonas syringae pv. syringae and Pseudomonas syringae pv. pisi , inoculation of susceptible cultivars was the most reliable. Results were confirmed by inoculation of lemon fruit.
A much more rapid and convenient serological method was developed to distinguish the two pathovars. Antisera against glutaraldehyde-fixed cells had a high level of specificity in Ouchterlony gel double-diffusion tests and, after cross-absorption with heterologous antigens, in indirect ELISA. Antiserum to P. syringae pv. pisi has considerable potential to detect pea seed infected with this pathogen.     

Identification of pathovars and races of <Emphasis Type="Italic">Pseudomonas syringae,</Emphasis> the main causal agent of bacterial disease in pea in North-Central Spain,and the search for disease resistance

A total of 298 bacterial isolates were collected from pea cultivars, landraces and breeding lines in North-Central Spain over several years. On the basis of biochemical-physiological characteristics and molecular markers, 225 of the isolates were identified as Pseudomonas syringae, either pv. pisi (110 isolates) or pv. syringae (112), indicating that pv. syringae is as frequent as pv. pisi as causal agent of bacterial diseases in pea. Most strains (222) were pathogenic on pea. Further race analyses of P. syringae pv. pisi strains identified race 4 (59.1% of the isolates of this pathovar), race 2 (20.0%), race 6 (11.8%), race 5 (3.6%) and race 3 (0.9%). Five isolates (4.6%) showed a not-previously described response pattern on tester pea genotypes, which suggests that an additional race 8 could be present in P. syringae pv. pisi. All the isolates of P. syringae pv. syringae were highly pathogenic when inoculated in the tester pea genotypes, and no significant pathogenic differences were observed. Simultaneous infections with P. syringae pv. pisi and pv. syringae in the same fields were observed, suggesting the importance of resistance to both pathovars in future commercial cultivars. The search for resistance among pea genotypes suitable for production in this part of Spain or as breeding material identified the presence of resistance genes for all P. syringae pv. pisi races except for race 6. The pea cultivars Kelvendon Wonder, Cherokee, Isard, Iceberg, Messire and Attika were found suitable sources of resistance to P. syringae pv. syringae.     

Influence of rootstock on the susceptibility of sweet cherry scions to bacterial canker, caused by Pseudomonas syringae pvs morsprunorum and syringae

In a field trial to determine whether the rootstock influenced the susceptibility of cherry cultivars to bacterial canker three cultivars (Napoleon, Roundel and JI 14039), each grafted on two rootstocks (F 12/1 and Colt), were subjected to natural infectionand to inoculation with three bacterial canker pathogens (Pseudomonas syringae pv. morsprunorum races 1 and 2 and P. syringae pv. syringae). Inoculations were made through leaf scars and through wounds. The high susceptibility of Napoleon and high resistance of JI 14039 were confirmed. Napoleon was more susceptible to inoculation through branches when on F12/1 than when on Colt but the reverse was true for leaf scar inoculations. JI14039 was more susceptible to race 1 inoculated through leaf scars when grown on F12/1 than when on Colt. No rootstock/scion interaction was detected with Roundel.
The complexity of the relationships between the pseudomonad pathogens and their cherry hosts is briefly discussed.     

Identification and origin of races of Pseudomonas syringae pv. phaseolicola from Africa and other bean growing areas

Isolates of Pseudomonas syringae pv. phaseolicola from Africa and other bean growing areas were categorized into nine races on the basis of their reactions to eight differential cultivars following artificial inoculation. Eight hundred and ninety-three isolates representing 303 disease occurrences were initially identified as P.s. pv. phaseolicola by their pathogenicity to bean, cultural and serological characteristics and phage sensitivity. These tests also served to distinguish P.s. pv. phaseolicola from the closely related pathovars P.s . pv. glycinea and P.s. pv. syringae . Detailed race determinations were carried out on 175 selected isolates of p.s. pv. phaseolicola representative of the different geographical regions and hosts in which the pathogen was found and nine races were identified. A number of races (1,2,5,6 and 7) were distributed worldwide with race 6 predominant. Other races were found mainly in Africa; races 3 and 4 in East/Central Africa and races 8 and 9 in Southern Africa. Most isolates were obtained from the major host, Phaseolus vulgaris . Alternative natural hosts included 10 legume species representative of seven different genera ( Cajanus cajan, Desmodium sp., Lablab purpureus, Macroptilium atropurpureum, Neonotonia wightii, Phaseolus acutifolius, P. coccineus, P. lunatus, Vigna angularis and V. radiata ). Of these, Desmodium sp. constitutes a new host record.     

Activation of systemic disease resistance in pea by an avirulent bacterium or a benzothiadiazole, but not by a fungal leaf spot pathogen

Inoculation of first expanded leaves of pea seedlings with an avirulent strain of Pseudomonas syringae pv. pisi , or treatment with sprays of a benzothiadiazole (20 or 100  μ g a.i. mL⁻¹), decreased the susceptibility of subsequent leaves 7 or 14 days later to challenge inoculation with Mycosphaerella pinodes . Inoculation of first leaves with a virulent strain of P. syringae pv. pisi or with M. pinodes did not decrease the susceptibility of plants to M. pinodes . Treatments effective in decreasing susceptibility to M. pinodes were similarly active against Uromyces viciae-fabae and virulent P. syringae pv. pisi . Effective treatments also enhanced the activities of the enzymes β-1,3-glucanase and chitinase in untreated upper leaves 6 days later. Ineffective treatments for decreased susceptibility had no effect on the activity of the enzymes. None of the treatments enhanced peroxidase activities. The results are discussed in relation to the reported signalling effects of the benzothiadiazole and in relation to a suggested high activity of the avirulent P. syringae pv. pisi strain and inactivity of M. pinodes in enhancing natural signalling.     

Inheritance of resistance to bacterial blight in 21 cultivars of rice

ABSTRACT Genetic analysis for resistance to bacterial blight (Xanthomonas oryzae pv. oryzae) of 21 rice (Oryza sativa L.) cultivars was carried out. These cultivars were divided into two groups based on their reactions to Philippine races of bacterial blight. Cultivars of group 1 were resistant to race 1 and those of group 2 were susceptible to race 1 but resistant to race 2. All the cultivars were crossed with TN1, which is susceptible to all the Philippine races of X. oryzae pv. oryzae. F(1) and F(2) populations of hybrids of group 1 cultivars were evaluated using race 1 and F(1) and F(2) populations of hybrids of group 2 cultivars were evaluated using race 2. All the cultivars showed monogenic inheritance of resistance. Allelic relationships of the genes were investigated by crossing these cultivars with different testers having single genes for resistance. Three cultivars have Xa4, another three have xa5, one has xa8, two have Xa3, eight have Xa10, and one has Xa4 as well as Xa10. Three cultivars have new, as yet undescribed, genes. Nep Bha Bong To has a new recessive gene for moderate resistance to races 1, 2, and 3 and resistance to race 5. This gene is designated xa26(t). Arai Raj has a dominant gene for resistance to race 2 which segregates independently of Xa10. This gene is designated as Xa27(t). Lota Sail has a recessive gene for resistance to race 2 which segregates independently of Xa10. This gene is designated as xa28(t).     

Comparison of Ethylene Production by Pseudomonas syringae and Ralstonia solanacearum

ABSTRACT Strains of Pseudomonas syringae pv. pisi and Ralstonia solanacearum produced ethylene at rates 20- and 200-fold lower, respectively, than strains of P. syringae pvs. cannabina, glycinea, phaseolicola, and sesami. In the current study, we investigated which ethylene biosynthetic pathways were used by P. syringae pv. pisi and R. solanacearum. Neither the activity of an ethylene-forming enzyme nor a corresponding efe gene homolog could be detected in R. solanacearum, suggesting synthesis of ethylene via 2-keto-4-methyl-thiobutyric acid. In contrast, 2-oxoglutarate-dependent ethylene formation was observed with P. syringae pv. pisi, and Southern blot hybridization revealed the presence of an efe homolog in this pathovar. The efe genes from P. syringae pvs. cannabina, glycinea, phaseolicola, pisi, and sesami were sequenced. Nucleotide sequence comparisons indicated that the efe gene in pv. pisi was not as highly conserved as it was in other P. syringae pathovars. The pv. pisi efe homolog showed numerous nucleotide substitutions and a deletion of 13 amino acids at the C-terminus of the predicted gene product. These sequence alterations might account for the lower rate of ethylene production by this pathovar. All ethylene-producing P. syringae pathovars were virulent on bush bean plants. The overlapping host range of these pathovars suggests that horizontal transfer of the efe gene may have occurred among bacteria inhabiting the same host.     

The occurrence of a new genetic variant of Fusarium oxysporum f.sp. pisi

During a survey of root diseases of pea in Denmark, a new genetic variant of Fusarium oxysporum f.sp. pisi was isolated from vining peas in two widely separated geographical regions. In terms of pathogenicity on a set of differential pea lines, the Danish isolated closely resembled a race 6 isolate from the United States, DNA extracts of the isolates, restricted with the endonuclease Hind III, then probed with a homologous repetitive genomic fragment from the plasmid pDG106 by the Southern hybridization technique, gave a unique'fingerprint'pattern distinctly different from the American race 6 and all other known races. When probed with pDG312, containing a homologous ribosomal repeat unit, the pattern obtained for the Danish isolates was indistinguishable from races 1, 5 and 6 but distinctly different from 2A and 2B. The Danish isolates represent a separate vegetative compatibility group because they are compatible with each other but incompatible with the other known races. In pigmentation the new variant resembled races 1, 5 and 6 for the first 8-12 days, after which it began to secrete a dark purple pigment resembling that of race 2A and 2B. Until an additional line in the host differentials can separate the new genetic variant it should be considered a subgroup of F. oxysporum f. sp. pisi race 6.     

豆薯细菌性角斑病的病原鉴定

 在安徽滁州的豆薯叶片上发现一种由细菌侵染引起角斑症状的病害,从角斑上分离到具有致病性的非荧光的杆状细菌,菌株的表型特征、细菌学特征、LOPAT试验和生理生化试验表明该细菌与丁香假单胞菌(Pseudomonas syringae van Hall)相似,BIOLOG系统鉴定结果与丁香假单胞菌豌豆致病变种(P.syringae pv.pisi)相近,接种试验表明豆薯菌株能侵染大豆、菜豆和眉豆,但对豌豆的致病性差;在豇豆、绿豆和蚕豆上不表现症状。结果表明豆薯细菌性角斑病是一种新病害,病原菌属于丁香假单胞菌群的一个新的致病变种,命名为P.syringae pv.pachyrhizus nov.     

Variation for virulence among Scandinavian isolates of Peronospora viciae f.sp. pisi (pea downy mildew) and responses of pea genotypes

Variation for virulence among Scandinavian isolates of Peronospora viciae f.sp. pisi (downy mildew) on different pea genotypes was investigated. Variation for virulence was found within and between mass-conidial isolates which were originally obtained from oospore populations. Virulence towards pea cultivars that have not been commerically cultivated in Scandinavia was observed. The specific resistance of the pea cultivars Puget, Cobri, Gastro, Starcovert and Starnain was confirmed. One pea breeding line showed a stable partial resistance to different isolates of the fungus and it was also highly resistant to race 8'from The Netherlands which is considered to be virulent on most pea genotypes carrying known specific resistance factors.     

Patterns of interaction between isolates of three pathovars of Pseudomonas syringae and accessions of a range of host and nonhost legume species

Isolates of three pathovars of Pseudomonas syringae were tested against 10 legume species. Some isolates of all pathovars showed cultivar-specific interactions with at least one legume species outside the expected host range. Lablab purpureus and Phaseolus lunatus were found to be hosts to isolates of both P. syringae pv. glycinea and P. syringae pv. phaseolicola, while Lathyrus latifolius was host to isolates of P. syringae pv. pisi and P. syringae pv. glycinea . Lens culinaris showed patterns of interaction with isolates of all three pathovars. Gene models based on mathematical estimates of minimum gene numbers agreed with those previously published for the interactions of P. syringae pv. pisi with Pisum sativum and P. syringae pv. phaseolicola with Phaseolus vulgaris. Two different gene-for-gene models based on five resistance/avirulence gene pairs were proposed to explain observed interactions between Glycine max and P. syringae pv . glycinea . Pathogen isolates which contained no known avirulences defined on their respective host species were found to carry cryptic avirulences recognized by other plant species. Estimates of minimum gene numbers required to explain the interactions of a plant species with all pathogen isolates or to explain the interactions of the isolates of one pathovar with all plant accessions were consistently lower than the sum of the minimum gene numbers required to explain the interactions of each individual component.     

Nontoxigenic Strains of Pseudomonas syringae pv. phaseolicola Are a Main Cause of Halo Blight of Beans in Spain and Escape Current Detection Methods

ABSTRACT From a collection of 152 pseudomonads isolated from diseased beans in Spain, 138 (91%) of the strains were identified as Pseudomonas syringae pv. phaseolicola and the rest as P. syringae pv. syringae. The P. syringae pv. phaseolicola strains produced typical water-soaked lesions on bean pods, although 95 of them did not produce phaseolotoxin in vitro. Ninety-four of these isolates did not produce the expected 0.5-kb product after polymerase chain reaction (PCR) amplification using primers specific for open reading frame (ORF) 6 of the phaseolotoxin (tox) gene cluster and did not contain DNA homologous to ORF 6 in Southern hybridization experiments. To our knowledge, this is the first report of the widespread occurrence in the field of strains of P. syringae pv. phaseolicola lacking the tox cluster, which contrasts sharply with the general belief that Tox(+) isolates are the only ones with epidemiological importance. Additionally, the tox(-) isolates were not specifically detected by a commercial polyclonal antisera in an enzyme-linked immunosorbent assay. Accordingly, it is possible that the certification of seed lots as free of the pathogen cannot be reliably done in Spain, or in any other country where tox(-) strains might occur frequently, using current PCR or serological protocols. The amplification of three avirulence genes by PCR allowed us to make predictions of the P. syringae pv. phaseolicola race structure, as confirmed by plant assays. Six races (races 1, 2, 5, 6, 7, and 9) were identified, with race 7 being the most prevalent (46.1%) followed by races 6 (21.3%) and 1 (9.0%). All the tox(-) isolates contained gene avrPphF, typical of races 1, 5, 7, and 9.     

Serological detection and identification of bacteria from plants by the conjugated Staphylococcus aureus slide agglutination test

A rapid slide agglutination test using polyclonal antisera conjugated to protein A-rich whole-cell Staphylococcus aureus was developed for the detection and identification of bacteria from plants. The specificity and sensitivity of the technique was evaluated in 18 antibody/antigen combinations, representing six bacterial genera ( Erwinia, Lactobacillus, Pseudomonas, Rhizobium, Rhodococcus and Xanthomonas ). For two pathovars of Pseudomonas syringae the specificity of the technique was increased by the use of antisera prepared to somatic extracts.
The advantages of the Staphylococcus aureus agglutination technique include speed, simplicity and the ability to identify organisms directly from infected plant tissues. It was applied to the detection of Pseudomonas syringae pv. phaseolicola and pv. pisi in lesions on bean and pea, respectively, to P. gladioli pv. alliicola and Lactobacillus sp. from rotted onion bulbs and specific strains of Rhizobium phaseoli in bean root nodules.     

Genetic,biochemical and pathogenic diversity of Pseudomonas syringae pv. pisi strains

Pseudomonas syringae pv. pisi is a seedborne pathogen distributed worldwide that causes pea bacterial blight. Previous characterization of this pathogen has been carried out with relatively small and/or geographically limited samples. Here, a collection of 91 strains are examined that include strains from recent outbreaks in Spain (53 strains) and from 14 other countries, and that represent all races and the new race 8, including the type race strains. This collection was characterized on the basis of 55 nutritional tests, genetic analysis (rep‐PCR, amplification of AN3 and AN7 specific markers, and multilocus sequence typing (MLST)) and pathogenicity on the differential pea cultivars to identify races. Principal component analysis and distance dendrograms confirm the existence of two genetic lineages within this pathovar, which are clearly discriminated by the AN3/AN7 markers, rep‐PCR and MLST. Strains from races 1 and 7 amplified the AN3 marker; those from races 2, 6 and 8 amplified AN7, while strains of races 3, 4 and 5 amplified either AN3 or AN7. Nevertheless, strains were not grouped by race type by any of the genetic or biochemical tests. Likewise, there was no significant association between metabolic and/or genetic profiling and the geographical origin of the strains. The Spanish collection diversity reflects the variability found in the worldwide collection, suggesting multiple introductions of the bacteria into Spain by contaminated seed lots.     

Effect of bacterial blight (Pseudomonas syringae pv. pisi) on the growth and yield of single pea (Pisum sativum) plants under glasshouse conditions

Single pea ( Pisum sativum ) plants cvs Kelvedon Wonder and Solara, growing in pots in a glasshouse, showed significant reductions in seed yield of 24, 47 or 71% following inoculation with pea bacterial blight ( Pseudomonas syringae pv. pisi ) during reproductive, vegetative or both reproductive and vegetative growth stages, respectively. These yield reductions were seen as reduced numbers of seeds per pod combined with changes in the numbers of pods per plant. Examination of the importance of different disease parameters on yield showed that mean disease on the whole plant was the most important. Equations describing the relationship between seed yield or the natural log of the total weight and mean disease were derived.     

Race-specific reaction of resistance to black rot in Brassica oleracea

Several black rot-resistant varieties of Brassica oleracea showed a race-specific hypersensitive response (HR) to inoculation with Xanthomonas campestris pv. campestris isolates of different races. In progenies of cabbage line PI436606, Portuguese kale ISA454 and Chinese kale SR1 the HR to race 1 of the pathogen was controlled by a dominant gene named R1, when a recessive gene r5 was responsible for the HR to race 5. Genes with a similar race-specific reaction were assumed on the basis of gene-for-gene interaction in black rot-resistant Japanese cabbage cultivars and double haploid lines obtained from them. Homology of gene r5 in cabbage lines PI436606, Fujiwase 01 and kale ISA454 was postulated in crosses between those lines or their progenies. In a cross between SR1 and PI436606, interaction between resistance to race 1 and non-specific resistance localized in the stem vascular system was found. On the basis of pedigree information and homology of resistance genes in the cultivars of East-Asian cabbage and Portuguese kales, the probable origin of race-specific resistance to black rot of cole crops was suggested to be in heading Mediterranean kale. Some evidence was found for a gene conferring resistance to race 4 in B. oleracea.     

Effect of soil moisture on the transmission of pea bacterial blight (Pseudomonas syringae pv. pisi) from seed to seedling

Controlled-environment studies in which pea seed cv. Solara, inoculated with Pseudomonas syringae pv. pisi , was sown in pots of compost maintained at different moisture contents showed that soil moisture had a considerable influence on the transmission of the disease from seed to seedling. An equation was derived from this data which described the relationship between the proportion of seedlings infected ( p ) and the soil water stress ( s ) in MPa: -In(-In(1 − p )) = 0.64 In( s ) + 4.5. This equation was used to produce predicted transmission rates for each year from 1987 to 1990, which were compared with measured transmission rates in field experiments at Wellesbourne in the same years. Although agreement between the observed and predicted transmission rates was poor, years of severe and slight disease transmission were successfully predicted.     

Identification of Isolates that Cause a Leaf Spot Disease of Brassicas as Xanthomonas campestris pv. raphani and Pathogenic and Genetic Comparison with Related Pathovars

ABSTRACT Twenty-five Xanthomonas isolates, including some isolates received as either X. campestris pv. armoraciae or pv. raphani, caused discrete leaf spot symptoms when spray-inoculated onto at least one Brassica oleracea cultivar. Twelve of these isolates and four other Xanthomonas isolates were spray- and pin-inoculated onto 21 different plant species/cultivars including horseradish (Armoracia rusticana), radish (Raphanus sativus), and tomato (Lycopersicon esculentum). The remaining 13 leaf spot isolates were spray-inoculated onto a subset of 10 plant species/cultivars. The leaf spot isolates were very aggressive on several Brassica spp., radish, and tomato causing leaf spots and dark sunken lesions on the middle vein, petiole, and stem. Based on the differential reactions of several Brassica spp. and radish cultivars, the leaf spot isolates were divided into three races, with races 1 and 3 predominating. A differential series was established to determine the race-type of isolates and a gene-for-gene model based on the interaction of two avirulence genes in the pathogen races and two matching resistance genes in the differential hosts is proposed. Repetitive-DNA polymerase chain reaction-based fingerprinting was used to assess the genetic diversity of the leaf spot isolates and isolates of closely related Xanthomonas pathovars. Although there was variability within each race, the leaf spot isolates were clustered separately from the X. campestris pv. campestris isolates. We propose that X. campestris isolates that cause a nonvascular leaf spot disease on Brassica spp. should be identified as pv. raphani and not pv. armoraciae. Race-type strains and a neopathotype strain for X. campestris pv. raphani are proposed.