Genetic relationship between races of Pseudomonas syringae pv.pisi and cultivars of Pisum sativum

Isolates of Pseudomonas syringae pv. pisi from the UK and overseas were categorized into six races on the basis of their reactions to a range of differential pea (Pisum sativum) cultivars. Race 2 was predominant among the isolates examined and this probably reflects its relative international importance. A previously uncharacterized race (race 6) was virulent on all cultivars tested. Resistance to races 1-5 was widespread in commercial cultivars and breeding lines with more than 75% showing resistance to one or more races. A preliminary study of the inheritance of resistance indicated that for races 1, 2 and 3, resistance was controlled by different dominant genes. The genetic basis for the relationship between races of P. syringae pv. pisi and pea cultivars was explained in terms of a gene-for-gene relationship involving five matching gene pairs. With further clarification of the genetics of resistance this host-pathogen association will meet most of the requirements of a model system for the study of the genetic and molecular basis of pathogenicity and host specificity.     

Erysiphe pisi var. pisi on faba beans and other legumes in Britain

The powdery mildew Erysiphe pisi var. pisi, complete with its teleomorph, has been observed on faba beans ( Vicia faba cultivars) for the first time in Britain. It appeared in a glasshouse on Syrian lines of fodder bean and English cultivars of broad bean. It also infected tare ( Vicia sativa ) and sweet pea ( Lathyrus odoratus ), but not edible pea ( Pisum sativum ), ornamental lupin ( Lupinus polyphyllus ), French bean ( Phaseolus vulgaris ) or sainfoin ( Onobrychis viciaefolia ). Inoculations in a glasshouse showed that another strain of E. pisi var. pisi found on outdoor everlasting pea ( Lathyrus latifolius ) was also capable of affecting faba bean, and was distinguished by its ability to infect edible pea. A third strain found on outdoor ornamental lupin infected only lupin. E. pisi var. pisi was shown to be morphologically and pathogenically distinct from the Erysiphe sp. newly affecting tomatoes in Britain.     

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.     

Disease assessment of pea lines with resistance to foot rot pathogens: protocols for in vitro selection

Cultural conditions were optimized so that disease reactions of plantlets and callus tissue from partially resistant and susceptible genotypes of Pisum sativum could be differentiated when inoculated with Fusarium solani f.sp. pisi. Disease was assessed using both semi-quantitative (disease score/fungal diameter) and quantitative methods (ergosterol assay).
After optimization of inoculum concentration and incubation period it was possible to differentiate the disease reactions of plantlets using disease scores and amount of ergosterol in the tissues.
Incubation period, temperature and the auxin added to the media were important in allowing differentiation of callus tissue cultures. These differences were visible by measurement of the diameter of fungal growth on the callus and the amount of ergosterol in the tissues, but not with disease scores.
The plantlet test was also applicable to the other foot rot pathogens Phoma medicaginis var. pinodella and Aphanomyces euteiches , allowing differentiation of the disease reactions of a partially resistant and susceptible genotype using disease scores. The ergosterol assay could be used to quantify infection caused by P. medicaginis var. pinodella , but this sterol was not detectable in mycelium of A. euteiches. In this fungus the presence of cholesterol was detected, which may be used to quantify infection.     

Host Specificity of Ascochyta spp. Infecting Legumes of the Viciae and Cicerae Tribes and Pathogenicity of an Interspecific Hybrid

ABSTRACT Ascochyta spp. (teleomorphs: Didymella spp.) infect a number of legumes, including many economically important species, and the diseases they cause represent serious limitations of legume production worldwide. Ascochyta rabiei, A. fabae, A. pisi, A. lentis, and A. viciae-villosae are pathogens of chickpea (Cicer arietinum), faba bean (Vicia faba), pea (Pisum sativum), lentil (Lens culinaris), and hairy vetch (V. villosa), respectively. Inoculations in the greenhouse and in growth chambers demonstrated that A. fabae, A. lentis, A. pisi, A. rabiei, and A. viciae-villosae were host specific. Isolates caused no visible disease symptoms on "nonhost" plants (plants other than the hosts they were originally isolated from) but were recovered consistently from inoculated, surface-disinfested, nonhost tissues. Interspecific crosses of A. pisi x A. fabae and A. viciae-villosae x A. lentis produced pseudothecia with viable ascospores, and the hybrid status of the ascospore progeny was verified by the segregation of mating type and amplified fragment length polymorphism (AFLP) markers. Interspecific progeny were morphologically normal in culture but exhibited more phenotypic variation compared with progeny from intraspecific crosses. Mating type and the majority of AFLP markers segregated in Mendelian 1:1 ratios in both intraspecific and interspecific crosses. A total of 11 and 7% of AFLP markers showed segregation distortion among progeny from interspecific crosses and intraspecific crosses, respectively; however, this difference was not significant (P = 0.90). Only 30 of 114 progeny isolates from the A. fabae x A. pisi cross inoculated in the greenhouse caused lesions on pea and only 4 caused disease on faba bean. In all, 15 of 110 progeny isolates were pathogenic to pea and none were pathogenic to faba bean under growth chamber conditions. Although no obvious postzygotic, intrinsic isolating barriers were identified in any of the interspecific crosses, it appears that host specialization may act as both a prezygotic, ecological isolating barrier and a postzygotic, extrinsic, ecological isolating barrier in these fungi. Host specificity, coupled with low pathogenic fitness of hybrids, may be an important speciation mechanism contributing to the maintenance of hostspecific, phylogenetic lineages of these fungi.     

Effect of powdery mildew (Erysiphe pisi) on nodulation and nitrogenase activity in pea (Pisum sativum)

The effects of powdery mildew ( Erysiphe pisi) disease of pea (Pisum sativum) on the number and size of root nodules and on nitrogenase activity were investigated in pea plants grown in pots and in the field. There were significant reductions in nodulation and in the size of root nodules as well as in nitrogenase activity in infected plants.     

Cytological Analysis of Defense-Related Mechanisms Induced in Pea Root Tissues in Response to Colonization by Nonpathogenic Fusarium oxysporum Fo47

The ability of nonpathogenic Fusarium oxysporum, strain Fo47, to trigger plant defense reactions was investigated using Ri T-DNA-transformed pea roots. Cytological investigations of strain Fo47-inoculated roots showed that the fungus grew actively at the root surface and colonized a number of epidermal and cortical cells, inducing marked host cell metabolic changes. In roots inoculated with pathogenic F. oxysporum f. sp. pisi, the pathogen multiplied abundantly through much of the tissues, whereas in Fo47-inoculated roots, fungal growth was restricted to the epidermis and the outer cortex. Invading cells of strain Fo47 suffered from serious alterations, a phenomenon that was not observed in control roots in which F. oxysporum f. sp. pisi grew so actively that the vascular stele was invaded within a few days. Strain Fo47 establishment in the root tissues resulted in a massive elaboration of hemispherical wall appositions and in the deposition of an electron-opaque material frequently encircling pathogen hyphae and accumulating in the noninfected xylem vessels. This suggests that the host roots were signaled to defend themselves through the rapid stimulation of a general cascade of nonspecific defense responses. The specific relationship established between strain Fo47 and the root tissues is discussed in relation to other types of plant-fungus interactions, including pathogenic and symbiotic associations.     

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.     

Development of monoclonal antibodies against the fungi of the 'Ascochyta complex'

Two monoclonal antibodies (MAbs) were produced against soluble antigens from the 'Ascochyta complex' fungi. Specificity of MAbs was tested by ELISA using antigen-coated wells. MAbs secreted by the monoclonal hybridoma cell line JIM 44 recognized epitopes present in the antigen preparations from Mycosphaerella pinodes and Phoma medicaginis var. pinodella , but not those present in preparations from Ascochyta pisi . At high tissue culture supernatant concentration, MAbs produced by the monoclonal line JIM 45 recognized epitopes from all three fungi, however, on dilution of MAb the antigens from A. pisi were recognized preferentially to those from M. pinodes and P. medicaginis var. pinodella . On the basis of heat and periodate treatment of the antigens from the three fungi it can be concluded that the epitope recognized by JIM 44 is carbohydrate in nature, whereas that recognized by JIM 45 is proteinaceous in nature, carried on a glycoprotein antigen. Antigen preparations from other fungi, including other pea pathogens, non-pathogens associated with pea and other fungi closely related to the 'Ascochyta complex', were not detected with either of the two MAbs. Antigen preparations from peas could be used to differentiate healthy and infected seeds in a dot-blot assay, therefore indicating the potential of using the MAbs in the development of a diagnostic test for infection of Pisum seeds by the 'Ascochyta complex' fungi.     

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.     

Toxicity of Glucosinolate Degradation Products from Brassica napus Seed Meal Toward Aphanomyces euteiches f. sp. pisi

ABSTRACT Brassica tissues are potentially useful in the control of Aphanomyces root rot of peas (Pisum sativum), but identity of the responsible compounds and specific impacts of those compounds on the pathogen's infection potential remain uncertain. Brassica napus seed meals and water extracts from these meals were used to determine the effect of glucosinolate hydrolysis products on Aphanomyces euteiches f. sp. pisi. B. napus meal ('Dwarf Essex') containing glucosinolates and intact myrosinase, the enzyme responsible for glucosinolate hydrolysis, completely inhibited infection by A. euteiches f. sp. pisi oospores. Water extracts from this meal, likewise, severely inhibited infection by oospores, as well as mycelial growth. Extracts from autoclaved 'Dwarf Essex' meal, in which myrosinase was denatured, and a low glucosinolate B. napus variety ('Stonewall') produced little disease reduction and had less impact on mycelial growth. Gas chromatographic analysis of Brassica tissues and water extracts confirmed that glucosinolates remained in autoclaved 'Dwarf Essex' meal and that 'Stonewall' meal contained low glucosinolate concentrations. 5-Vinyloxazolidine-2-thione was identified by mass spectrometry as a dominant glucosinolate hydrolysis product in aqueous extracts of the inhibitory meal. Bioassays conducted with aqueous solutions of this compound reduced mycelial growth, but not to the extent of those from intact 'Dwarf Essex' meal. Water-soluble compounds produced from the hydrolysis of glucosinolates in B. napus tissues reduced A. euteiches oospore infection and inhibited mycelial growth, thus, demonstrating potential utility of Brassica species in the control of A. euteiches.     

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.     

Biological Control of Pathogens Causing Root Rot Complex in Field Pea Using Clonostachys rosea Strain ACM941

ABSTRACT Pea root rot complex (PRRC), caused by Alternaria alternata, Aphanomyces euteiches, Fusarium oxysporum f. sp. pisi, F. solani f. sp. pisi, Mycosphaerella pinodes, Pythium spp., Rhizoctonia solani, and Sclerotinia sclerotiorum, is a major yield-limiting factor for field pea production in Canada. A strain of Clonostachys rosea (syn. Gliocladium roseum), ACM941 (ATCC 74447), was identified as a mycoparasite against these pathogens. When grown near the pathogen, ACM941 often was stimulated to produce lateral branches that grew directly toward the pathogen mycelium, typically entwining around the pathogen mycelium. When applied to the seed, ACM941 propagated in the rhizosphere and colonized the seed coat, hypocotyl, and roots as the plant developed and grew. ACM941 significantly reduced the recovery of all fungal pathogens from infected seed, increased in vitro seed germination by 44% and seedling emergence by 22%, and reduced root rot severity by 76%. The effects were similar to those of thiram fungicide, which increased germination and emergence by 33 and 29%, respectively, and reduced root rot severity by 65%. When soil was inoculated with selected PRRC pathogens in a controlled environment, seed treatment with ACM941 significantly increased emergence by 26, 38, 28, 13, and 21% for F. oxysporum f. sp. pisi, F. solani f. sp. pisi, M. pinodes, R. solani, and S. sclerotiorum, respectively. Under field conditions from 1995 to 1997, ACM941 increased emergence by 17, 23, 22, 13, and 18% and yield by 15, 6, 28, 6, and 19% for the five respective pathogens. The seed treatment effects of ACM941 on these PRRC pathogens were greater or statistically equivalent to those achieved with thiram. Results of this study suggest that ACM941 is an effective bioagent in controlling PRRC and is an alternative to existing chemical products.     

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.     

An ultrastructural study of tomato roots inoculated with pathogenic and non-pathogenic necrotrophic fungi and a saprophytic fungus

Tomato cultivar Moneymaker was independently inoculated with Alternaria alternata, Cunninghamella elegans, Fusarium culmorum, F. oxysporum f.sp. lycopersici, F. oxysporum f.sp. pisi and Stromatinia gladioli and analysed ultrastructurally. The extent and amount of superficial fungal growth on tomato roots was similar but C. elegans , a saprophyte, was exceptional in that hyphae were not closely appressed to plant surfaces and did not adhere to plant cell walls.
In general, the type of plant responses to fungal colonization and infection were similar in all of the interactions studied, with the exception of C. elegans which did not infect tomato root tissue. The failure to penetrate tomato roots by C. elegans may have been associated with the lack of hyphal adhesion to plant cell walls. Migration of cytoplasm and wall apposition/penetration papilla formation were regularly observed in tomato root tissue beneath appressed hyphae and at sites of fungal infection. Specific cellular reactions in the exodermis, namely the formation of wall 'inclusions' and appearance of 'sensitive' cells, indicated that exodermal cells were particularly responsive to fungal challenge.
Fusarium oxysporum f.sp. lycopersici , a pathogen of tomato, invaded tomato root tissue more extensively than the other fungi inoculated onto tomato roots. Infection of tomato by the other fungi studied was variable, and the extent and success of fungal invasion was tentatively associated with their necrotrophic capability and typical host range.     

用生物间遗传学研究水稻品种和稻瘟病菌之间的相互关系

在温室中,将供试的13个水稻品种和7个鉴别品种的幼苗用稻瘟病菌的11个菌株分别进行接种,以同等条件下鉴别品种对菌株的反应所确定的小种,作为供试品种对该小种的反应,然后应用生物间遗传学的原理和方法来推导品种和菌株的基因型。结果表明:供试的水稻品种和稻瘟病菌株(或小种)间存在17个限定性相应基因对,不同品种所含的抗病性基因数有所不同,其作用是在相应的无毒性菌株(或小种)存在情况下而表现出品种的抗病性基因愈多,可能表现的抗病力愈强。本研究结果为水稻抗稻瘟病育种选择抗原亲本以及生产上选用具有较多抗病基因的品种进行合理布局,提供了有重要参考价值的遗传学信息和依据。     

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.     

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.     

Development of a Scale for Evaluation of Tomato yellow leaf curl virus Resistance Level in Tomato Plants

ABSTRACT We have developed a scale of differential hosts that enables the determination and comparison of level of resistance to Tomato yellow leaf curl virus (TYLCV) expressed by resistant tomato lines or by individual plants in a segregating population. The scale is composed of seven different homozygous tomato genotypes that exhibit different levels of TYLCV resistance, ranging from fully susceptible to highly resistant. The differential hosts composing the scale were inoculated with TYLCV under greenhouse conditions. Four weeks after inoculation the plants were evaluated for disease symptom severity, and virus DNA titer was determined. The different genotypes were arranged in the scale according to symptom severity score. The different genotypes were then tested under different environmental conditions, inoculated at different ages, and tested in a field experiment assaying TYLCV-induced yield reduction. While the symptom severity score of each individual resistant genotype changed under different environmental conditions, the relative position on the scale did not alter, except for one genotype. Thus, to evaluate disease resistance of a given tomato genotype, the genotype in question should be inoculated alongside the differential hosts composing the scale, and within 4 weeks one can determine the relative level of resistance of the tested genotype.