Regulation of Polyphosphoinositide Metabolism in Pea Plasma Membranes by Elicitor and Suppressor from a Pea Pathogen, Mycosphaerella pinodes

Effects of the elicitor and the suppressor from a pea pathogen,Mycosphaerella pinodes, on polyphosphoinositide metabolism inpea plasma membranes were examined in vitro. Lipid phosphorylationin the isolated pea plasma membrane was drastically stimulatedby the elicitor, but markedly inhibited by the suppressor. Asimilar inhibitory effect was observed by the treatment withorthovanadate or K-252a that blocked pisatin production inducedby the elicitor. Neomycin, an aminoglycoside antibiotic thatinteracts with the polyphosphoinositide metabolism, also affectedthe lipid phosphorylation in vitro and blocked the elicitor-inducedaccumulation of pisatin in vivo. These results suggest thatrapid changes of polyphosphoinositide metabolism in pea plasmamembranes is one of indispensable processes during the elicitationof defense responses. (Received January 22, 1992; Accepted March 23, 1992)     

Inhibition of ATPase Activity in Pea Plasma Membranes In Situ by a Suppressor from a Pea Pathogen, Mycosphaerella pinodes

A pathogenic fungus of pea, Mycosphaerella pinodes, secretesa so-called "suppressor" in its pycnospore germination fluid.The suppressor blocks the defense responses and induces localsusceptibility (accessibility) in pea plants to agents thatare not pathogenic in pea. The suppressor nonspecifically inhibitsthe ATPase activity in plasma membranes prepared from pea, soybean,kidney bean, cowpea and barley plants. However, cytochemicalstudies by electron microscopy indicate that the suppressorspecifically inhibits the ATPase in pea cell membranes, butnot in those of four other plant species tested. That is, thespecificity of the suppressor appears at the cell and/or tissuelevel, but is not evident in vitro. Furthermore, the inhibitoryeffect of the suppressor is temporary because the ATPase activityrecovers 9 h after the treatment. A similar effect was observedafter inoculation with M. pinodes but not with a nonpathogenof pea, M. ligulicola. The role of the suppressor in host-parasitespecificity is discussed. (Received April 9, 1991; Accepted August 6, 1991)     

Isolation and Characterization of Double-Stranded RNAs from the Pea Pathogen, Mycosphaerella pinodes

Studies on the structure of the extrachromosomal nucleic acidsin the plant pathogenic fungus, Mycosphaerella pinodes, OMP-1,isolated from Okayama, Japan, indicate that at least three double-stranded(ds) RNAs are present in the cytoplasm, while a strongly pathogenicisolate from England, MP-3, does not contain any of the dsRNAs.The sizes of these dsRNAs are 6.0 kb (L), 4.0 kb (M), and 2.6kb (S), respectively. Weakly pathogenic mutants selected fromUV-irradiated OMP-1 contain considerably higher copy numberof S than does the parental strain, while one of the mutants,OMP-X62, contains no detectable M. OMP-1 was cured of detectabledsRNAs by treatment with cycloheximide. Curing was accompaniedby an increase in virulence and by partial recovery of a colonymorphology similar to that of MP-3. The presence of these dsRNAsapparently results in a reduction in mycelial growth, in thestimulation of sporulation and possibly in the promotion ofhypovirulence. (Received February 19, 1990; Accepted October 30, 1990)     

Inhibition of ATPase Activity in Pea Plasma Membranes by Fungal Suppressors from Mycosphaerella pinodes and Their Peptide Moieties

The effects of two suppressors of the defense reactions of hostplants, which had been purified from the pea pathogen Mycosphaerellapinodes, as well as the effects of peptide moieties, on theATPase activity in pea plasma membranes were examined in vitro.One of the suppressors, Supprescin B, inhibited the ATPase activityin a non-competitive manner, but the other suppressor, SupprescinA, did not. Supprescin A was observed to reduce the inhibitoryeffect of Supprescin B. A tripeptide, Ser-Ser-Gly, and a hexapeptide,Ser-Ser-Gly-Asp-Glu-Thr, which were the respective peptide moietiesof Supprescin A and B, inhibited the ATPase activity in a competitivemanner. Supprescin B and fragments of the hexapeptide, suchas Asp-Glu-Thr and Gly- Asp-Glu, inhibited not only the ATPaseactivity but also the acid phosphatase activity of plasma membranesin vitro. These results indicate that the acidic amino-acidresidues of the "Asp-Glu" moiety seem to act as inhibitors ofthe phosphatase activity. Thus, the peptide moiety of SupprescinB consists of at least two functional elements. (Received October 23, 1992; Accepted January 18, 1993)     

Suppression of Pisatin Production and ATPase Activity in Pea Plasma Membranes by Orthovanadate, Verapamil and a Suppressor from Mycosphaerella pinodes

A pea pathogen, Mycosphaerella pinodes, secretes both an elicitorand a suppressor for the accumulation of pisatin, a major phytoalexinof pea, into the spore germination fluid. The effects of theelicitor and the suppressor on the ATPase activity in pea plasmamembranes was examined. The ATPase was sensitive to orthovanadateand dicyclohexylcarbodiimide but insensitive to nitrate andazide; it was unaffected by the elicitor but was markedly inhibitedby the suppressor (50µg.ml^–1, bovine serum albuminequivalents) or verapamil (1OOµM). The accumulation ofpisatin induced by the elicitor was delayed for 3 to 6 h inthe presence of orthovanadate or verapamil to an extent similarto that in the presence of the suppressor. The relationshipbetween the inhibition of plasma membrane ATPase activity andthe suppression of the active defense reaction that involvesthe production of pisatin in the pea plant is discussed. (Received April 16, 1990; Accepted September 6, 1990)     

A Pathotype Classification for Mycosphaerella pinodes

Genetic variation of Mycosphaerella pinodes in the pathogenesis of Pisum sativum is described for the first time. On a particular host line, isolates varied from those producing a few necrotic flecks to those causing large lesions on stems or leaves. Based on reactions of nine differential host lines, 45 isolates from a wide range of geographical locations could be classed, by stem symptoms, into 9 groups or, by leaf symptoms, into 16 groups.     

Pea seed infection by Mycosphaerella pinodes and Ascochyta pisi and its control by seed soaks in thiram and captan suspensions

Apparently normal pea seeds from pods bearing lesions of Mycosphaerella pinodes were often internally infected with the fungus. When infected seeds were sown in sterile grit almost all the seedlings showed disease lesions, at or below soil level, 4–6 weeks after sowing. Seed infected with Ascochyta pisi gave only 40% infection of seedlings: these showed lesions on the stem and first two leaves within 4 weeks of sowing. Infection of seeds by both pathogens could be eradicated by soaking the seeds for 24 hr. in 0.2% suspensions of thiram or captan at 30d?C. In laboratory or greenhouse tests these treatments did not check germination, but in the field the captan treatment reduced emergence. The treated seeds became fully imbibed but could be dried and stored: the thiram treatment was used for semi-commercial treatment of quantities of seed up to 3 cwt.     

Leaf Spots Induced by Ascochyta pisi and Mycosphaerella pinodes

The leaf-spot pathogens, Ascochyta pisi and Mycosphaerella pinodes,both cause limited, necrotic lesions in detached pea leafletssuspended above water. When leaflets were floated on water A.pisi lesions were still limited, but those caused by M. pinodesspread rapidly to occupy all the leaflet tissue. Increasing the numbers of spores in inocula decreased numbersof mature lesions caused by A. pisi, but increased numbers ofspreading lesions caused by M. pinodes. Older leaflets weremore susceptible to both pathogens. Studies of penetration and colonization of leaves with the aidof light and electron microscopy showed that cell-wall-degradingenzymes were involved in the formation of A. pisi lesions andin spreading lesions caused by M. pinodes. There was littleevidence of cell-wall degradation in limited M. pinodes lesionsin which penetration of walls by hyphae seemed to be mechanicalin nature. No physical barriers developed in tissues surrounding limitedlesions. Nevertheless, A. pisi was rarely found beyond the necroticarea. This suggested that tissues beyond the lesion had becomeresistant to the parasite. In contrast, M. pinodes often grewoutside the necrotic area, sometimes many days after this hadstopped growing, but when it did so it caused no more necrosisunless leaflets were placed in conditions in which the spreadingtype of lesion could develop.     

Orthovanadate Suppresses Accumulation of Phenylalanine Ammonia-Lyase mRNA and Chalcone Synthase mRNA in Pea Epicotyls Induced by Elicitor from Mycosphaerella pinodes

Orthovanadate delayed accumulation of mRNAs encoding phenylalanineammonia-lyase and chalcone synthase in pea epicotyls inducedby an elicitor from Mycosphaerella pinodes. However, accumulationof mRNA for a putative P-type ATPase was not affected. The relationshipbetweenthe ATPase and defense responses is discussed. ³Present address: Plant Pathology Laboratory, School of Agriculture,Nagoya University, Chikusa, Nagoya, 464-01 Japan.     

Non-Specific Induction of Pisatin and Local Resistance in Pea Leaves by Elicitors from Mycosphaerella pinodes, M. melonis and M. ligulicola and the Effect of Suppressor from M. pinodes

The accumulation of pisatin was induced non-specifically by elicitors prepared from the high molecular weight fraction (molecular weight: more than 10,000 daltons) of the spore germination fluid of three species of Mycosphaerella-plant pathogens in pea leaves with epidermis removed, regardless of the pathogenicity of the fungi to pea. Before the elicitation of pisatin synthesis, local resistance to infection by Mycosphaerella pinodes was induced by elicitors again non-specifically inpea leaves in which wax had been removed from the leaf surface. The substance responsible for local resistance could be extracted with ethylacetate from the elicitor-containing drop diffusate which was placed on pea leaves. The substance prevented the penetration of M. pinodes through heat-killed pea epidermis, but did not affect spore germination. The suppressor prepared from the low molecular weight fraction (molecular weight: less than 10,000 daltons) of the spore germination fluid of M.pinodes counteracted the ability of elicitors to induce both phases of resistance mechanisms.     

Studies on the survival of Mycosphaerella pinodes and Ascochyta pisi

Laboratory and field experiments on two pea-pod and leaf-spot pathogens, Mycosphaerella pinodes and Ascochyta pisi, have indicated that the former species may survive overwinter in soil whether introduced as pycnidiospores, mycelium, chlamydospores or sclerotia. The survival of pycnidiospores appears to be due at least in part to their transformation into chlamydospores in the soil. A. pisi was much less successful in soil; the mycelium inoculum survived, somewhat sporadically, and spore viability was reduced, while the total number of spores declined markedly. Investigations on the competitive saprophytic ability of these pathogens showed that by the agar plate method M. pinodes could be classed as a moderately successful saprophyte, though the Butler and Park methods indicated only intermediate and poor success respectively. A. pisi was adjudged a poor saprophyte by all three methods. Saprophytic growth and survival in soil of both fungi was generally better at 2 °C than at 15 °C.     

In vivo control of Mycosphaerella pinodes on pea leaves by a crude bulb extract of Eucomis autumnalis

We previously reported on the in vitro antifungal activity of a crude whole plant extract from Eucomis autumnalis against seven economically important plant pathogenic fungi. A crude extract of the bulb showed similar in vitro mycelial growth inhibition of the same plant pathogenic fungi as well as that of an eighth fungus, Mycosphaerella pinodes, the cause of black spot or Ascochyta blight, in peas. Subsequently, fourth internode leaves were removed from 4 wk old pea plants, placed on moist filter paper in Petri dishes and inoculated with an M. pinodes spore suspension before and after treatment with the extract. The control of Ascochyta blight by different concentrations of the crude E. autumnalis extract was followed in vivo by leaf symptoms over a 6 day period at 20°C in a growth cabinet. The crude extract prevented M. pinodes spore infection of the leaves when the leaves were inoculated with spores both before or after treatment with the extract, confirming complete inhibition of spore germination. The crude E. autumnalis extract showed no phytotoxic reaction on the leaves even at the highest concentration applied.     

Role of Cell-wall-degrading Enzymes in the Development of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

Extracts of limited and spreading lesions caused by Mycosphaerellapinodes on detached pea leaflets contained proteolytic, cellulolytic,and pectolytic enzymes although only in spreading lesions wasthere much degradation of cell walls. The brown tissue fromlimited M. pinodes lesions was resistant to maceration by enzymesfrom spreading lesions. Limited lesions contained water-soluble,95 per cent ethanol insoluble, partially dialysable, inhibitorsof pectin transeliminase which is probably the macerating enzyme. Green, spreading M. pinodes lesions developed only on leafletsfloating on water. Growth of these lesions was accompanied bycontinous loss of phenolic substances to the water while thephenol content in infected tissue remained similar to that inuninoculated controls. In contrast, the phenol content in mature,limited M. pinodes lesions on leaflets suspended just abovethe water level was about four times that in healthy tissue.It is suggested that loss of phenolics from floating leafletsprevents tissue browning and the development of resistance ofthe cell walls to maceration. But this type of resistance doesnot appear to be a major factor in the limitation of lesionson suspended tissue. Extracts of limited Ascochyta pisi lesions on leaflets floatingon water contained pectolytic and hemicellulolytic enzymes.Some cellulase (Cx) activity was detected although there waslittle evidence of cellulose degradation in cell walls in infectedtissue. The nature of the macerating factor remains uncertainbut it was found that extracts from lesions contained inhibitorsof pectic enzymes and that tissue just beyond that colonizedby the fungus was resistant to maceration; this resistance isprobably important in restricting the growth of the pathogenin the leaf.     

Effects of pod infection by Mycosphaerella pinodes on yield components of pea (Pisum sativum)

Yield reduction of pea (Pisum sativum) due to various types of infections by Mycosphaerella pinodes on pods was assessed. A range of disease severities was created on pods of pea plants grown in the glasshouse, by painting the pods with different concentrations of spore suspensions, at three different pod development stages: lag phase, the beginning of seed filling (BSF) and mid-filling of the seeds. Seed number at harvest was reduced only if the pods were infected before BSF, as shown previously for whole plant infections. Pod infections led to individual seed weight (ISW) losses from zero (for late infections, at mid-filling) to 20% (for earlier infections and severe disease). Infection during the lag phase affected ISW by reducing seed growth rate, whereas infection at BSF tended to reduce the duration of seed filling. There was a linear relationship between the area under the disease progress curve and the percentage decrease in ISW. This model should be complemented by the effect of leaves and stem infections, in order to predict ISW losses in diseased crop conditions, in which epidemics occur on all aerial parts of the pea plant.     

A Single,Plastic Population of Mycosphaerella pinodes Causes Ascochyta Blight on Winter and Spring Peas (Pisum sativum) in France

Plant diseases are caused by pathogen populations continuously subjected to evolutionary forces (genetic flow, selection, and recombination). Ascochyta blight, caused by Mycosphaerella pinodes, is one of the most damaging necrotrophic pathogens of field peas worldwide. In France, both winter and spring peas are cultivated. Although these crops overlap by about 4 months (March to June), primary Ascochyta blight infections are not synchronous on the two crops. This suggests that the disease could be due to two different M. pinodes populations, specialized on either winter or spring pea. To test this hypothesis, 144 pathogen isolates were collected in the field during the winter and spring growing seasons in Rennes (western France), and all the isolates were genotyped using amplified fragment length polymorphism (AFLP) markers. Furthermore, the pathogenicities of 33 isolates randomly chosen within the collection were tested on four pea genotypes (2 winter and 2 spring types) grown under three climatic regimes, simulating winter, late winter, and spring conditions. M. pinodes isolates from winter and spring peas were genetically polymorphic but not differentiated according to the type of cultivars. Isolates from winter pea were more pathogenic than isolates from spring pea on hosts raised under winter conditions, while isolates from spring pea were more pathogenic than those from winter pea on plants raised under spring conditions. These results show that disease developed on winter and spring peas was initiated by a single population of M. pinodes whose pathogenicity is a plastic trait modulated by the physiological status of the host plant.     

Role of Inhibitors of Fungal Growth in the Limitation of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

The phytoalexin, pisatin, was detected in host tissues 24 hafter inoculation of pea leaflets with spores of the leaf-spottingpathogens Ascochyta pisi and Mycosphaerella pinodes. Pisatincontinued to accumulate in infected tissue as A. pisi lesionsdeveloped and was present in inhibitory concentrations in thebrown tissue beyond the region colonized by the pathogen. During the formation of limited M. pinodes lesions, concentrationsof pisatin were highest 2 days after inoculation. Levels weremore variable and lower in older lesions which appeared to containno other inhibitors of germ-tube growth. Spreading lesions causedby M. pinodes on leaflets floating on water contained littleor no pisatin although little was released to the water below.These lesions did, however, contain other highly active inhibitorsof germ-tube growth. The significance of these results in terms of limitation oflesions are discussed. The ease with which M. pinodes lesionscan become progressive may reflect the greater ability of thispathogen to grow in high concentrations of pisatin under certainconditions.     

Characterization of the metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas (Pisum sativum L.)

The metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas, was detected by thin layer chromatography (TLC) and was analyzed for its chemical and pathogenic characteristics. One blue dot was detected using 254nm UV light on TLC plate, and a spray of rho-anisaldehyde (110 degrees C, 30 min) also produced a blue dot. The solvent systems used for TLC analysis were ethyl acetate/water/acetone (5/2/5), chloroform/methanol/glacial acetic acid (19/10/2), toluene/ethyl acetate/90% formic acid (6/3/1), diethylether/methanol/water/90% formic acid (95/4/1/1), and bezene/methanol/acetic acid (24/2/1), with R(f) values (min-max) of 0.09-0.18, 0.88-0.95, 0.06-0.15, 0.39-0.47 and 0.05-0.12, respectively. The recovered metabolite from the TLC plate displayed UV absorption peaks at 212, 244, 250, 256 and 261 nm. The proposed formula of the main component of the metabolite was C16H12N3O6. The TLC-purified metabolite induced symptom of discoloration on detached pea leaves.     

Induction of defense responses by synthetic glycopeptides that have a partial structure of the elicitor in the spore germination fluid of Mycosphaerella pinodes

A high molecular weight elicitor (> 70 kDa) from spore germination fluid of a pea pathogen, Mycosphaerella pinodes, has a partial structure of beta-D-Glc-(1-->6)-alpha-D-Man-(1-->6)-D-Man, which is O-glycosidically attached to serine in the protein moiety. To elucidate the minimum structure for the elicitor activity to pea plants, the effects of nine glycopeptides including beta-D-Glc-(1-->6)-alpha-D-Man-(1-->6)-D-Man-O-Ser (No. 1) to [beta-D-Glc-(1-->6)-alpha-D-Man-(1-->6)-D-Man]3-O-Ser3-Pro3 (No. 9) on the infection by M. pinodes, superoxide generation and ATPase activity were measured. The glycopeptides [beta-D-Glc-(1-->6)-alpha-D-Man-(1-->6)-D-Man]-O-Ser2-Pro2 (No. 3) to No. 9 induced rejection reaction of pea tissue against M. pinodes. The glycopeptides No. 3 to No. 9 also induced superoxide generation on uninjured pea leaves. Moreover, the glycopeptides No. 3 to No. 9 induced in vitro the activation of cell wall-bound ATPase and superoxide generation system in the protein fraction solubilized from pea cell wall. The results indicate that the synthetic glycopeptides, No. 3 to No. 9, are available to analyze the signal transduction cascade leading to defense responses and the receptor for the elicitor.