Inheritance of resistance to <Emphasis Type="Italic">Mycosphaerella pinodes</Emphasis> in two wild accessions of <Emphasis Type="Italic">Pisum</Emphasis>

Mycosphaerella pinodes is one of the most devastating pea pathogens. Pea cultivars with adequate levels of resistance to control the disease are not so far available. However, promising levels of resistance have been identified in wild accessions of pea. In the present investigation the inheritance of resistance to M. pinodes was studied in two crosses between the susceptible pea cv. ‘Ballet’ and the partially wild resistant accessions P665 (Pisum sativum subsp. syriacum) and P42 (P. sativum subsp. sativum var. arvense). Both additive and dominant effects were important in control of resistance and susceptibility dominated over resistance.     

Identification of genes expressed during spore germination of Mycosphaerella pinodes

Mycosphaerella blight, caused by Mycosphaerella pinodes, is one of the major diseases of cultivated pea (Pisum sativum L.). To isolate the genes that are up- and down-regulated during spore germination, suppression subtraction hybridization (SSH) was performed between ungerminated and germinated spores. The 232 and 128 clones from forward and reverse libraries, respectively, were collected, sequenced, and analyzed with a BLASTX homology search. About 95% of the 32 selected clones were expressed during spore germination on a paper sheet and during infection of pea leaves. We discuss the applicability of the SSH libraries for analyzing M. pinodes genes involved in the early stage of infection.     

Differential regulation of MBP kinases by a glycoproptein elicitor and a polypeptide suppressor from Mycosphaerella pinodes in pea

A polypeptide fungal suppressor from a pea pathogen Mycosphaerella pinodes plays a key role in pathogenesis by suppressing elicitor-induced defense response(s) in pea (Pisum sativum L). In this study, we show that treatment of pea tissues with the polysaccharide elicitor secreted by M. pinodes results in rapid increased activation of two myelin basic protein (MBP)-dependent kinases p44 (≈44 kDa) and p48 (≈48 kDa) within 15–30 min upon elicitation. Interestingly, the suppressor inhibited the elicitor-induced activation of only p44 kinase. While the defense-inducing signalling molecules, chitosan and salicylic acid (SA) activated the p44 and p48 kinases, methyl jasmonate (MeJA) did not. The abiotic stress signals, abscisic acid (ABA), NaCl and wounding activated the p48 kinase alone. These results demonstrate that MAPKs are differentially activated in response to pathogen invasion and abiotic stress in pea. Furthermore, specific inhibition of elicitor-induced p44 kinase activation by a MAPKK inhibitor, PD098059 and protein kinase inhibitor, K252a correlated with the suppression of elicitor-induced phenylalanine ammonia lyase (PAL) gene expression, supporting a role for p44 in the elicitor-induced defense response(s) in pea. Inhibition of p44 by the phosphoinositide (PI) turnover inhibitor, neomycin (a fungal suppressor mimic), and potentiation of p44 by the diacylglycerol (DAG) kinase inhibitor, R59022 indicated that p44 may be acting downstream of (PI) metabolism. Taken together, our results indicate that suppressor of defense elicitation from M. pinodes acts through inhibition of a MAPK (p44), possibly through a PI signaling pathway, facilitating the establishment of basic compatibility during infection of pea.     

Host status and reaction of <Emphasis Type="Italic">Medicago truncatula</Emphasis> accessions to infection by three major pathogens of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>)

Ditylenchus dipsaci, the stem nematode of alfalfa (Medicago sativa), Mycosphaerella pinodes, cause of Ascochyta blight in pea (Pisum sativum) and Aphanomyces euteiches, cause of pea root rot, result in major yield losses in French alfalfa and pea crops. These diseases are difficult to control and the partial resistances currently available are not effective enough. Medicago truncatula, the barrel medic, is the legume model for genetic studies, which should lead to the identification and characterization of new resistance genes for pathogens. We evaluated a collection of 34 accessions of M. truncatula and nine accessions from three other species (two from M. italica, six from M. littoralis and one from M. polymorpha) for resistance to these three major diseases. We developed screening tests, including standard host references, for each pathogen. Most of the accessions tested were resistant to D. dipsaci, with only three accessions classified as susceptible. A very high level of resistance to M. pinodes was observed among the accessions, none of which was susceptible to this pathogen. Conversely, a high level of variation, from resistant to susceptible accessions, was identified in response to infection by A. euteiches.     

A binding protein for fungal signal molecules in the cell wall of Pisum sativum

In the plant cell wall of Pisum sativum seedlings, we found an NTPase (E.C. 3.6.1.5.) with ATP-hydrolyzing activity that was regulated by an elicitor and suppressors of defense from pea pathogen Mycosphaerella pinodes. The ATPase-rich fraction was purified from pea cell walls by NaCl solubilization, ammonium sulfate precipitation, and chromatography with an ATP-conjugated agarose column and an anion-exchange column. The specific activity of the final ATPase-rich fraction increased 600-fold over that of the initial NaCl-solubilized fraction. The purified ATPase-rich fraction also had peroxidase activity and generated superoxide, both of which were regulated by the M. pinodes elicitor and suppressor (supprescins). Active staining and Western blot analysis also showed that the ATPase was copurified along with peroxidases. In this fraction, a biotinylated elicitor and the supprescins were bound primarily and specifically to ca. 55-kDa protein (CWP-55) with an N-terminal amino acid sequence of QEEISSYAVVFDA. The cDNA clone of CWP-55 contained five ACR domains, which are conserved in the apyrases (NTPases), and the protein is identical to a pea NTPase cDNA (GenBank accession AB071369). Based on these results, we discuss a role for the plant cell wall in recognizing exogenous signal molecules.     

Cloning and characterization of pea apyrases: involvement of <Emphasis Type="Italic">PsAPY1 </Emphasis>in response to signal molecules from the pea pathogen <Emphasis Type="Italic">Mycosphaerella pinodes</Emphasis>

 Two nucleoside triphosphatase (NTPase) cDNA clones were isolated from a cDNA library of Pisum sativum L., cv. Midoriusui. The genes encoding the cDNAs were designated PsAPY1 and PsAPY2. PsAPY1 included the N-terminal amino acid sequence of an NTPase bound to pea cell wall. The phylogenic analysis indicated that PsAPY1 belongs to an NTPase subfamily responsive to environmental stimuli and that PsAPY2 belongs to a discrete subfamily, the physiological role of which is almost unknown. The adenosine triphosphatase activity of recombinant PsAPY1 was regulated by an elicitor and a suppressor from the pea pathogen Mycosphaerella pinodes. Based on these findings, we discuss the role of NTPases in response to biological stresses. Received: May 27, 2002 / Accepted: July 31, 2002     

Plant cell walls as suppliers of potassium and sodium ions for induced resistance in pea (Pisum sativum L.) and cowpea (Vigna unguiculata L.)

When an elicitor is applied to plants to induce resistance, one of the first detectable events is the efflux of ions from the treated tissue. Here we are the first to demonstrate that an elicitor from Mycosphaerella pinodes evokes leakage of Na⁺ and K⁺ ions from isolated cell walls of pea and cowpea in vitro, as observed for epicotyl tissues. Pharmacological experiments showed that this elicitor-stimulated leakage was sensitive to vanadate and N-(3-methylphenyl)biphenyl-4-sulfonamide (NGXT-191), that inhibit a cell wall-associated ATPase (apyrase). Vanadate or NGXT-191 suppressed elicitor-induced superoxide generation and expression of defense genes in vivo. On the basis of these results, we assume that the leakage of these ions, probably associated with an ATP-dependent process(es) in the cell wall, is likely associated with induced defenses of pea and cowpea.     

Zur Verbreitung und Bedeutung von Pilzkrankheiten in K?rnerfuttererbsen (Pisum sativum L.) in Deutschland

Monitoring of the pathogen spectrum in grain peas (Pisum sativum L.) was conducted in Germany between 2005 and 2007. The outcome of this study implies that the infections of pathogens depend on the annual weather condition and the geographic area.Ascochyta pinodes, which infects aerial plant organs, was the pathogen found most frequently (on average 61, 8%). Especially in years with moist weather conditions a more severe spreading of the pathogen could be observed. Similarly, the occurrence ofBotrytis cinerea depends on the weather conditions. In 2007 were optimal conditions for infections and consequentlyB. cinerea was found with a high frequency of about 70%. Regarding diseases on root and stem base, this study clearly shows that infection of less prominent Fusarium species, such asF. redolens andF. avenaceum was significantly higher compared toF. oxysporum andF. solani.     

Expression of defence‐related genes in stems and leaves of resistant and susceptible field pea (Pisum sativum) during infection by Phoma koolunga

The differential expression of 13 defence‐related genes during Phoma koolunga infection of stems and leaves of susceptible versus resistant field pea (Pisum sativum) was determined using qRT‐PCR. Expression, in terms of relative mRNA level ratios, of genes encoding ferredoxin NADP oxidoreductase, 6a‐hydroxymaackiain methyltransferase (hmm6), chalcone synthase (PSCHS3) and ascorbate peroxidase in leaves and stems differed during 6–72 hours post‐inoculation (hpi) and reflected known host resistance levels in leaves versus stems. In comparison to the susceptible genotype, at 24, 48 and 72 hpi, two genes, hmm6 (122.43‐, 206.99‐ and 32.25‐fold, respectively) and PSCHS3 (175.00‐, 250.13‐ and 216.24‐fold, respectively), were strongly up‐regulated in leaves of the resistant genotype, highlighting that resistance against P. koolunga in field pea is governed by the early synthesis of pisatin. At 24 hpi, leaves infected by P. koolunga showed clear differences in expression of target genes. For example, the gene encoding a precursor of the defensin ‘disease resistance response protein 39’ was substantially down‐regulated in leaves of both the susceptible and the resistant genotypes inoculated with P. koolunga. This contrasts with other studies on another pea black spot pathogen, Didymella pinodes, where this same gene is strongly up‐regulated in leaves of resistant and susceptible genotypes. The current study provides the first understanding of defence‐related genes involved in the resistance against P. koolunga, opening novel avenues to engineer new field pea cultivars with improved leaf and stem black spot disease resistance as the basis for developing more effective and sustainable management strategies.     

Resistance in field pea (Pisum sativum) to the black spot disease complex in Western Australia

Black spot (also referred as Ascochyta blight, Ascochyta foot rot and black stem; Ascochyta leaf and pod spot) is a devastating disease of field pea (Pisum sativum) caused by one or more pathogenic fungi including Didymella pinodes, Ascochyta pisi, Phoma pinodella and P. koolunga. Development of resistant germplasm has been slow because of the low level of resistance found in the available germplasm, poor reliability of screening methods and the polygenic nature of inheritance. Field studies, undertaken to assess F8 and F9 derived lines for resistance against the black spot complex, confirmed that some lines developed in the Australian breeding program show improvement in resistance over commercial cultivars. Disease scores across test lines ranged from 5.33 to 7.82 (0–9 scale where 0?=?no disease symptoms, and 9?≥?90 % leaf area affected) and from 5.37 to 8 in 2012 and 2013, respectively. In 2012, the eight most resistant lines had scores 5.33 to 6, with OZP1207 the most resistant line. In 2012, forty three lines were significantly more resistant (disease score?≤?6.67) than the susceptible standard, Helena (7.82), 14 lines were not significantly different to the most resistant commercial cultivar, Parafield (6.33), and 27 lines were significantly more resistant than PBA Percy (7.67). In 2013, WAPEA2211 was clearly the most resistant line (5.37) followed by a group of seven lines with slightly less resistance scored at 6. Use of these lines in breeding will further enhance resistance in commercial cultivars, particularly by inter-crossing among the more genetically diverse lines to accumulate minor genes for resistance. While there was no overall relationship between disease scores in 2012 and 2013 (R ²?=?0.029), presumably due to the highly variable pathogen composition of the black spot complex at the screening site and across seasons in Western Australia, a few lines, such as WAPEA2211, 04H349P-05HO2005, 06H109P-9 and 06H459P-1, showed significant resistance in both years, appear to have resistance to multiple pathogens in the black spot complex, and are of particular significance.     

Imazethapyr inhibition of acetolactate synthase in Rhizobium and its symbiosis with pea

Acetolactate synthase (ALS) activity extracted from Rhizobium leguminosarum biovar. viciae has been characterized. The optimum pH for extraction was 7·6 and for the assay 7·0. The K_m for pyruvate was 7·2 mM , and the enzyme was saturated at 40 mM . An obligatory requirement of TPP and Mg²⁺ for full ALS activity was observed. Valine was the only branched-chain amino acid that caused ALS feedback inhibition. The specific activity of Rhizobium ALS was nearly 20 times the activity found in pea (Pisum sativum) leaves. Bacteroids from pea nodules also showed high ALS activity, and the nodule plant fraction had higher ALS activity than other plant tissues. ALS sensitivity to imazethapyr was also dependent on the source: ALS activity of free-living Rhizobium and bacteroids was slightly more tolerant than that of other pea tissues, but the differences were less than those found in rates of specific activity. It is proposed that the high ALS activity expressed by Rhizobium, both as free-living bacteria and as bacteroids, is related to the growth tolerance of rhizobia to imazethapyr and is also related to the relative tolerance of symbiotic pea plants. © 1998 SCI     

Identification and characterization of pathogens associated with root rot of winter peas grown under organic management in Germany

Root rots are limiting factor for pea production worldwide. This disease is caused by a pathogen complex and the role of single pathogens is unclear. This study aimed at identifying pathogens involved in a root rot of organically grown field pea in Germany, and establishing their importance in the disease complex. The potential of yard waste compost to suppress the diseased was also studied. Average disease severity index was similar in 2010 and 2011 (DI of 4.56 to 4.59, respectively) but it increased in 2012 to DI 5.8. Peyronellaea pinodella was most frequently isolated pathogen, with isolation frequency from 86%, 73% and 86% in 2010, 2011 and 2012, respectively. In addition, Didymella pinodes, Fusarium solani f. sp. pisi, F. oxysporum f. sp. pisi and F. avenaceum were the main fungi recovered from pea roots. In pathogenicity test all of the tested pathogens caused weak symptoms on the pigmented winter variety EFB33 and moderate to severe symptoms on the white flowering summer variety Santana. F. avenaceum was the most aggressive pathogen on Santana with DI of 7.4 followed by P. pinodella with DI of 5.7. The high aggressiveness combined with the wide host range highlights the possibility of F. avenaceum emerging as potential risk for organic crop rotation. High levels of resistance of EFB33 against all pathogens shows the potential of this variety to serve as a resource in further research for identification and development of new sources of resistance against root rot diseases of pea.     

H2O2 production by copper amine oxidase, a component of the ecto-apyrase (ATPase)-containing protein complex(es) in the pea cell wall, is regulated by an elicitor and a suppressor from Mycosphaerella pinodes

Blue native PAGE analysis for cell wall proteins from pea epicotyls demonstrated that cell wall-associated ecto-apyrase (ATPase) formed a large protein complex(es) ranging from 450 to 900?kDa; one of the components of the complex was copper amine oxidase (CuAO), which catalyzes the oxidation of amines with the subsequent generation of ammonia and hydrogen peroxide. CuAO activity was coordinately regulated in vitro with ATP-hydrolyzing activity by an elicitor and a suppressor from Mycosphaerella pinodes. Moreover, treatment of cell wall proteins with the suppressor caused the appearance of the apyrase monomer. On the basis of these results, M. pinodes may target the apyrase-containing protein complex(es) of the host to attenuate cell wall-based, extracellular defense(s) including the production of hydrogen peroxide.     

Suppression of mRNAs for lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase (OPR) in pea reduces sensitivity to the phytotoxin coronatine and disease development by Mycosphaerella pinodes

Using a recently developed model pathosystem involving Medicago truncatula and Mycosphaerella pinodes, causal agent of Mycosphaerella blight on pea to understand host molecular response to a fungal suppressor, we applied the suppressor to leaves of M. truncatula and identified 151 nonredundant cDNA fragments as newly expressed genes. These included genes encoding lipoxygenase (LOX) and enoyl-CoA hydratase, which are presumably involved in jasmonic acid (JA) synthesis. Potential genes encoding plastidic enzymes, including allene oxide synthase (AOS) and allene oxide cyclase (AOC), and other peroxisomal enzymes involved in β-oxidation were predicted from the Medicago Gene Index EST database and tested for altered expression by semiquantitative RT-PCR. The coordinated expression of genes encoding both plastidic and peroxisomal enzymes showed that the suppressor likely conditions certain cellular process(es) through the JA synthesis in M. truncatula. To explore the role of JA or JA-regulated cellular process(es) in conditioning susceptibility, we used an Apple latent spherical virus (ALSV)-based virus-induced gene silencing (VIGS) technology to silence pea genes including LOX, AOS, AOC and 12-oxo-phytodienoic acid reductase (OPR). In LOX-, AOS-, AOC- or OPR-silenced pea plants, disease development induced by M. pinodes was remarkably reduced. Similarly, silencing of mRNA for LOX, AOS, AOC or OPR reduced the sensitivity to a phytotoxin, coronatine, which is believed to act through a JA-dependent process. On the basis of these results, it is conceivable that M. pinodes has evolved a strategy to condition susceptibility by manipulating the physiology of host cells, in particular JA-regulated cellular process(es), to promote disease development in pea.     

Spatiotemporal distribution of Ascochyta pinodes and Ascochyta pinodella during the winter growing season in France

Ascochyta blight of pea is caused by four related fungi, Ascochyta pisi, Phoma koolunga, Ascochyta pinodes and Ascochyta pinodella. The latter two taxa appear to be much more common and economically significant worldwide but the relative impact of each fungus on ascochyta blight epidemics is not well understood. To study the spatiotemporal distribution of A. pinodes and A. pinodella infecting pea in France, 368 isolates were sampled monthly, from February to May, at three locations (Rennes, Boigneville and Dijon) and molecular markers were used to genotype isolates. The aggressiveness of isolates from the fourth sampling date was estimated using a detached leaf assay on the winter cultivar Enduro. Disease was low during the sampling period as climatic conditions were generally not conducive to disease development (cold temperature, low rainfall). Population genetic analysis showed that 99% of the observed variation could be attributed to variation within populations compared to only 1% among populations. Both species were observed in each location, although A. pinodella was observed at a lower frequency (6–32%). Moreover, results showed that both species could develop on different nodes of the plant. Significant differences in aggressiveness were observed between species and among isolates within species with A. pinodes isolates being significantly more aggressive on average than A.  pinodella isolates. These results emphasize the necessity to study the components of disease complexes in order to understand the impact of pathogen species interactions on disease and yield reduction as well as the dynamics of disease epidemics during the cropping season.     

Relationship between ascochyta blight on field pea (Pisum sativum) and spore release patterns of Didymella pinodes and other causal agents of ascochyta blight

Ascochyta blight of field pea, caused by Didymella pinodes, Phoma medicaginis var. pinodella, Phoma koolunga and Didymella pisi, is controlled through manipulating sowing dates to avoid ascospores of D. pinodes, and by field selection and foliar fungicides. This study investigated the relationship between number of ascospores of D. pinodes at sowing and disease intensity at crop maturity. Field pea stubble infested with ascochyta blight from one site was exposed to ambient conditions at two sites, repeated in 2 years. Three batches of stubble with varying degrees of infection were exposed at one site, repeated in 3 years. Every 2 weeks, stubble samples were retrieved, wetted and placed in a wind tunnel and up to 2500 ascospores g^?1 h^?1 were released. Secondary inoculum, monitored using seedling field peas as trap plants in canopies arising from three sowing dates and external to field pea canopies, was greatest in early sown crops. A model was developed to calculate the effective number of ascospores using predictions from G1 blackspot manager (Salam et al., 2011b; Australasian Plant Pathology, 40 , 621–31), distance from infested stubble (Salam et al., 2011a; Australasian Plant Pathology, 40 , 640–7) and winter rainfall. Maximum disease intensity was predicted based on the calculated number of effective ascospores, soilborne inoculum and spring rainfall over two seasons. Predictions were validated in the third season with data from field trials and commercial crops. A threshold amount of ascospores of D. pinodes, 294 g^?1 stubble h^?1, was identified, above which disease did not increase. Below this threshold there was a linear relationship between ascospore number and maximum disease intensity.     

Pea extracellular Cu/Zn-superoxide dismutase responsive to signal molecules from a fungal pathogen

We previously reported that the release of O₂ ⁻ from isolated pea cell walls was enhanced by a 70-kDa glycoprotein elicitor but was suppressed by mucin-type glycopeptide suppressors (supprescins A and B) prepared from pycnospore germination fluid of Mycosphaerella pinodes, causal agent of Mycosphaerella blight of pea. Here, we show that superoxide dismutase (SOD) in the apoplast fluid/cell wall of pea seedlings responds to the fungal elicitor and suppressor molecules. In a pharmacological study and with internal amino acid sequencing, the apoplastic SOD in a pea cultivar Midoriusui was found to be a Cu/Zn type SOD. We cloned a full-length cDNA of the Cu/Zn-SOD and designated it as PsCu/Zn-SOD1. An increase in PsCu/Zn-SOD1 mRNA and the PsCu/Zn-SOD1 protein was induced by treatment with the elicitor more intensively than by wounding. Such induction by the elicitor or wounding, however, was inhibited by the concomitant presence of supprescins. The SOD activity of recombinant PsCu/Zn-SOD1 was regulated directly by these signal molecules in a manner similar to their effect on the SOD activity in the apoplastic fluid and in the cell wall-bound proteins. Based on these findings, we discuss a role for PsCu/Zn-SOD1 in the pea defense response. The nucleotide sequence data of PsCu/Zn-SOD1 reported are available in the DDBJ/EMBL/GenBank databases under accession number AB189165.     

Biotic factors affecting the expression of partial resistance in pea to ascochyta blight in a detached stipule assay

The expression of partial resistance in pea to ascochyta blight (caused by Mycosphaerella pinodes) was studied in a detached stipule assay by quantifying two resistance components (fleck coalescence and lesion expansion) using the method of point inoculation of stipules. Factors determining optimal conditions for the observation of partial resistance are spore concentration, the age of the fungal culture prior to spore harvest and the pathogenicity of the isolate used for testing. Partial resistance was not expressed when spore concentration was high or when the selected isolate was aggressive. Furthermore, assessments of components of partial resistance were highly correlated with disease severity in a seedling test. A screening protocol was developed based on inoculations of detached stipules to study partial resistance in pea. To simplify the rating process, a more comprehensive disease rating scale which took into account fleck coalescence and lesion expansion was tested by screening a large number of genotypes.     

Orobanche crenata resistance and avoidance in pea (Pisum spp.) operate at different developmental stages of the parasite

Orobanche crenata (broomrape) is an important constraint to pea (Pisum sativum) cultivation in the Mediterranean area, because little resistance is available in commercial crop varieties. Field experiments have demonstrated that some resistance is present in a number of P. sativum and P. fulvum accessions. The goal of this work was to characterize such resistance. The Pisum–O. crenata interaction and the resistance symptoms were studied under controlled conditions by using Petri dish and polyethylene bag assays. The content of phenolics and peroxidase activity in host tissue from infected and non-infected plants were also measured. Resistance and avoidance mechanisms, acting at different developmental stages of the parasite, have been identified, including low stimulation of O. crenata seed germination, unsuccessful penetration of host roots, delay in post-attachment tubercle development and necrosis of the attached tubercles. Infection caused an increase in the content of total soluble phenolics in some Pisum genotypes. Peroxidase activity was higher in resistant than in susceptible accessions. Results obtained with different Pisum genotypes showed that resistance is the result of several mechanisms acting at different stages of the infection process. Resistance is also related to increased levels of peroxidase activity in host roots.