Endo-polygalacturonase from Cladosporium cucumerinum elicits lignification in cucumber hypocotyls

The cucumber pathogen Cladosporium cucumerinum produces one endo-polygalacturonase and at least two exo-polygalacturonases during growth in a liquid medium containing citrus pectin as the carbon source. The endo-polygalacturonase was purified nine-fold by ion-exchange chrumatography and gel filtration. The enzyme elicits lignification in cucumber hypocotyls down to a concentration of about 0·08 units ml⁻¹ which corresponds to about 70 ng protein ml⁻¹. It also releases elicitors of lignification from polygalacturonic acid and cucumber cell walls. The enzyme has a pH-optimum between 5·0 and 5·5, a molecular weight of about 38 000 and contains neutral hexose and protein in a ratio of 15:85 (w/w). The endo-polygalacturonase elicits lignification equally effectively in susceptible as in resistant cucumber hypocotyl segments, and releases about the same amount of elicitor from the cell walls of resistant and susceptible cucumber hypocotyls. This does not exclude the involvement of endo-polygalacturonase in the lignification reaction of resistant cucumber plants towards C. cucumerinum, but the specificity of the reaction must apparently be determined by other molecules.     

In vitro Effect of the Phytoalexin Glyceollin I on Polygalacturonase and Oxalic Acid Production ofSclerotinia sclerotiorum

The polygalacturonase (PG) activity in culture filtrates fromSclerotinia sclerotiorum is reduced when glyceollin I, the major soybean phytoalexin, is present in the culture medium. When the enzyme activities in the culture filtrates are expressed per unit of fungal growth, PG activity decreases with increasing concentration of glyceollin I in the culture medium. The phytoalexin does not influence the isoenzyme pattern. This suggests that glyceollin I may inhibit quantitative but not qualitative enzyme production. Only the highest glyceollin I concentration tested inhibits oxalic acid production. The inhibitory effect on mycelial growth is confirmed. The data suggest a further hypothesis about the role of phytoalexin during pathogenesis.     

Accumulation of sorghum phytoalexins induced by Colletotrichum graminicola at the infection site

Microspectrophotometry was performed on intact, pigmented vesicle-like inclusions within living sorghum cells that were accumulating phytoalexins as a response to attempted fungal infection. The results indicate that the deoxyanthocyanidin phytoalexins are present in inclusions. Moreover, the phytoalexin concentration within a single inclusion, based on luteolinidin, was calculated to be 0·15 m. The amounts of luteolinidin and apigeninidin in cells involved in the phytoalexin response at individual infection sites were also determined. The data showed that luteolinidin accumulated to levels of 0·48–1·20 ng/cell whereas apigeninidin accumulated to levels of 0·24–0·91 ng/cell. The results of both analyses confirmed that at the infection site the deoxyanthocyanidins accumulate to levels in substantial excess of those required for inhibition of the fungus Colletotrichum graminicola.     

The basis of host recognition in Fusarium oxysporum f. sp. lycopersici

Filtrates from shake-cultures of Fusarium oxysporum f. sp. lycopersici race 1, concentrated to 20% of the original volume, caused cell death in tomato leaf protoplasts from near-isogenic lines corresponding to the compatible cultivar/race reactions of whole plants. Maximum activity was found in late log phase cultures on Czapek-Dox supplemented with 2% casamino acids. Selective toxicity was associated only with the protein fraction of the culture filtrate. LD₅₀ values for susceptible Ace and Moneycross to F. oxysporum f. sp. lycopersici race 1 culture filtrates were 1·92 and 0·36 μg protein ml⁻¹. Corresponding values for cvs Royal Ace and MM161, each containing the I-gene conferring resistance to race 1, were >350. Culture filtrates from F. oxysporum f. sp. lycopersici race 2 gave LD₅₀ values of 2·34 and 2·08 μg protein ml⁻¹ on cvs Ace and Royal Ace, both susceptible to race 2. The LD⁵⁰ of cv. Ace to a non-pathogenic isolate of F. xysporum f. sp. lycopersici was > 350. Culture filtrates from non-host formae of F. oxysporum were 9–149-fold less toxic on cv. Ace. Protoplasts from Pisum sativum, Lactuca sativa, Zea mays, Gossypium barbadense and Solanum melongena, all non-hosts of F. oxysporum f. sp. lycopersici, were 6–175 times less sensitive to F. oxysporum f. sp. lycopersici filtrates than susceptible tomato. The putative toxins lycomarasmin and fusaric acid showed no differential toxicity to I⁺ and I⁻ tomato protoplasts. The results are discussed in the wider context of host-pathogen interaction in which specificity is considered as the recognition of susceptibility by a proteinaceous toxic metabolite of the pathogen. This hypothesis is further extended to include the specificity of F. oxysporum formae and races.     

Temperature-induced susceptibility of soybeans to Phytophthora megasperma f.sp. glycinea: phenylalanine ammonia-lyase and glyceollin in the host; growth and glyceollin I sensitivity of the pathogen

In unwounded soybean hypocotyls, pulse labelled with [¹⁴C]phenylalanine and inoculated with Phytophthora megasperma f.sp. glycinea, rates of [¹⁴C]-incorporation and glyceollin I accumulation were higher in resistant than in susceptible responses throughout the time-course of the experiment. This distinction was masked in hypocotyls that were wounded and inoculated. In such hypocotyls, high rates of [¹⁴C]-incorporation developed that were similar for the first 11 h in resistant and susceptible responses, although much more glyceollin I accumulated in the former. High rates of [¹⁴C]-incorporation also developed in uninoculated wounded hypocotyls but only small amounts of glyceollin I of high specific radioactivity were detected. Estimates of phenylalanine ammonia-lyase activity indicated that the metabolic flux through phenylalanine was limited in wounded controls but potentially very high in resistant responses. Differences in rates of [¹⁴C]-incorporation and in specific radioactivity of accumulated glyceollin I presumably indicate differences in the relative contributions of mobile internal pools and externally applied phenylalanine, in addition to rates of biosynthesis. Rapid decline in [¹⁴C]-glyceollin I was demonstrated in wounded controls in pulse-ch0ase experiments with phenylalanine as chase, but not in inoculated hypocotyls, due to continued [¹⁴C]-incorporation during the chase period. Rapid metabolism was demonstrated in all interactions and in wounds when cinnamic acid was used as the chase, but there was no evidence that differences in glyceollin I accumulation were due to differential rates of metabolism. Additional evidence for metabolic activity was provided by pulse feeding with [¹⁴C]glyceollin I. It is concluded that the stimulus of wounding or infection induces a metabolic pathway in which glyceollin I is not an end product. The accumulation of higher levels of glyceollin I in resistant than in susceptible responses appears to be due to earlier initiation and subsequently higher rates of biosynthesis in the former.     

An improved bioassay for victorin based on the use of oat protoplasts

A new bioassay for the host selective toxin from Helminthosporium victoriae was developed from studies of the effects on protoplasts. A suspension of protoplasts (0·2 ml) from susceptible or resistant oat leaves was placed in each well of microtiter plates and toxin (50 μl) was added. After incubation at 35 °C, protoplast death caused by toxin was determined by microscopic observation; quantitative data were possible by use of fluorescein diacetate as a vital strain. Toxin at 3·0 ng ml⁻¹ killed 50% of the protoplasts in 3 h, and 10 ng ml⁻¹ killed 90%. No toxin-induced death was observed in 20 min, even with very high toxin concentrations; thereafter, the percentage of dead cells increased with increased exposure time. Protoplasts were exposed to several toxin concentrations for 1, 10 and 30 min, then washed and incubated without toxin for 3 h; there were high, intermediate and minimal numbers of survivors, respectively. Survival percentages were equal in wells containing 2000 to 20 000 protoplasts, but were higher in wells containing > 20 000. Protoplasts from resistant oat leaves were not affected by toxin. The protoplast assay, which can be completed in 6 h, overcomes several limitations of other assays. The protoplast assay was used to test for toxin in culture filtrates and in solutions at all steps during the purification procedure.     

Quantitative Aspects of Infection of Sclerotinia sclerotiorum Sclerotia by Coniothyrium minitans – Timing of Application, Concentration and Quality of Conidial Suspension of the Mycoparasite

White mould disease leads to production of sclerotia, which subsequently survive in soil and may be responsible for future epidemics. The effect of the mycoparasite Coniothyrium minitans in decreasing survival of sclerotia of Sclerotinia sclerotiorum was studied. Infection of sclerotia of S. sclerotiorum by C. minitans can be achieved by a single conidium. Under optimal conditions, 2 conidia per sclerotium produced 63% of the maximum infection (ca. 90%) of sclerotia produced by up to 1000 conidia. Similar results were observed on the infection of stem pieces infected by S. sclerotiorum. In field trials, application of conidial suspensions of C. minitans to a bean crop soon after white mould outbreak led to a higher percentage of sclerotial infection than later applications. Ninety per cent infection of sclerotia was obtained within 3 weeks of application by C. minitans suspensions in the range of 5 × 10⁵ and 5 × 10⁶ conidia ml^–1 at 1000 l ha^–1. The concentration of the conidial suspensions and the isolate used were of less importance. The result was marginally affected by the germinability of the conidia (75% against 61% infected sclerotia at 91% and 16% viability of isolate IVT1, respectively). Less apothecia of S. sclerotiorum developed in soil samples collected after 2 months from plots sprayed immediately after disease outbreak than from those treated 11–18 days later. It is concluded that a suspension of 10⁶ conidia ml^–1 in 1000 l ha^–1 (= 10¹² conidia ha^–1) sprayed immediately after the first symptoms of disease are observed, results in > 90% infection of sclerotia of S. sclerotiorum. The infection of sclerotia, which prevents their carry-over, occurs within a broad range of inoculum quality.     

Accumulation of phytoalexins in susceptible and resistant near-isogenic lines of tomato infected with Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici

The time course of accumulation of two phytoalexins, the terpenoid rishitin and the polyacetylene cis-tetradeca-6-ene-1,3-diyne-5,8-diol, was determined in near-isogenic susceptible and resistant tomato lines inoculated with either Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici.Cultivars containing the Ve gene for verticillium wilt resistance accumulated phytoalexins at a rate similar to that in susceptible plants following stem inoculation with V. albo-atrum. Higher amounts of phytoalexins were isolated from susceptible than from resistant plants at 11 days after inoculation. Inoculum concentrations of 10⁵, 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ had no differential effect on phytoalexin accumulation at 3 days after inoculation. Also, no differences were observed between fungal growth in susceptible and resistant cultivars during that period.A cultivar containing the I-1 gene for fusarium wilt resistance contained more rishitin than did susceptible plants at 2 and 3 days after inoculation with 10⁷ conidia of F. oxysporum f.sp. lycopersici ml⁻¹, but at 7 and 11 days after inoculation more rishitin had accumulated in the susceptible plants.No difference was observed between the rate of accumulation of phytoalexin in stem segments from resistant and susceptible plants inoculated by vacuum-infiltration.To estimate the concentration of phytoalexins in the xylem fluid, sap was expressed from vascular tissue and amounts of phytoalexins were determined in the sap and in the expressed tissue. Less than 5% of the phytoalexins present in stem segments was recovered from the sap, indicating that their concentration in the xylem fluid may be relatively low.The role of phytoalexins in resistance to verticillium and fusarium wilt is discussed.     

Induction of oxidants in tomato leaves treated with <Emphasis Type="SmallCaps">DL</Emphasis>-β-Amino butyric acid (BABA) and infected with <Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> ssp. <Emphasis Type="Italic">michiganensis</Emphasis>

Bacterial canker is an economically important disease of tomato. Resistance induced by DL-β-Amino butyric acid against bacterial canker caused by Clavibacter michiganensis ssp. michiganensis in tomato plants was investigated. Different doses of DL-β-Amino butyric acid (250–1000 μg ml⁻¹ doses) were tested on 3-week old plants inoculated with a 10⁸ CFU ml⁻¹ bacterial suspension, and disease development was evaluated after inoculation and treatment. Although in vitro growth of the bacteria was not affected by DL-β-Amino butyric acid treatment, foliage sprays of 500 μg ml⁻¹ DL-β-Amino butyric acid significantly suppressed disease development up to 54% by day 14 after inoculation at the four different doses tested. Bacterial populations were reduced by 84% in BABA-treated plants compared to water-treated plants by day 4 after inoculation. Inoculated BABA-treated plants showed significantly higher phenylalanine ammonia-lyase activity, peroxidase activity, and H₂O₂ concentration than inoculated water-treated plants during day 1 after treatment. These findings suggest that the DL-β-Amino butyric acid treatment resulted in an increase of these enzymes and in H₂O₂ concentration in planta, and was associated with induction of resistance to bacterial canker.     

Purification of polygalacturonases produced by the pear scab pathogens, Venturia nashicola and Venturia pirina

An exopolygalacturonase and three endopolygalacturonases were purified from mycelia of pear scab pathogens, Venturia pirina and Venturia nashicola. The molecular weight of the isolated exoPG from V. pirina was 43 kDa, and the endoPGs from V. nashicola were 42 kDa as estimated by SDS–polyacrylamide gel electrophoresis. The pH optimum of the exoPG activity from V. pirina was 5.0. TheK_m and V_maxvalues of the exoPG were 0.08 mg ml⁻¹and 4.44 × 10⁻³ mmol reducing group min⁻¹ mg protein⁻¹. The N-terminal amino acid sequence of the exoPG from V. pirina was similar to that of the exoPG from Fusarium oxysporum f. sp. melonis, and the N-terminal amino acid sequences of the three endoPGs fromV. nashicola races 1, 2 and 3 were similar to other fungal endoPGs with a conserved motif of ASxxxTFTxAAAxxxG.     

Destruxin B produced by Alternaria brassicae does not induce accessibility of host plants to fungal invasion

It has been reported that Alternaria brassicae, the causal agent of gray leaf spot in Brassica plants, produces a host-specific or host-selective toxin (HSTs) identified as destruxin B. In this study, the role of destruxin B in infection of the pathogen was investigated. Destruxin B purified from culture filtrates (CFs) of A. brassicae induced chlorosis on host leaves at 50–100 μg ml⁻¹, and chlorosis or necrosis on non-host leaves at 250–500 μg ml⁻¹. Destruxin B was detected in spore germination fluids (SGFs) on host and non-host leaves, but not in a sufficient amount to exert toxicity to host plants. When spores of non-pathogenic A. alternata were combined with destruxin B at 100 μg ml⁻¹ and inoculated on the leaves, destruxin B did not affect the infection behavior of the spores. Interestingly, SGF on host leaves allowed non-pathogenic spores to colonize host leaves. Moreover, a high molecular weight fraction (>5 kDa) without destruxin B obtained by ultrafiltration of SGF had host-specific toxin activity and infection-inducing activity. From these results, we conclude that destruxin B is not a HST and does not induce the accessibility of the host plant which is essential for colonization of the pathogen. In addition, the results with SGF imply that a high molecular weight HST(s) is involved in the host–pathogen interaction.     

Induction of hypericins and hyperforin in Hypericum perforatum L. in response to biotic and chemical elicitors

Hypericum perforatum L. produces hyperforins, a family of antimicrobial acylphloroglucinols; and hypericins, a family of phototoxic anthraquinones exhibiting anti-microbial, anti-viral, and anti-herbivore properties in vitro. To determine whether these secondary metabolites are part of the specific plant defense systems that are mediated by methyl jasmonate or salicylic acid, we used meristem cultures to assess the effects of exposure to exogenous application of these chemical elicitors. Levels of hypericins in plant tissue increased in response to both elicitor treatments; total hypericin levels increased as much as 3.3 times control levels when treated with 200 μ methyl jasmonate for 14 days. Increased hyperforin concentrations were detected when plantlets were treated with 1 m salicylic acid or 50 μ methyl jasmonate. For assessing responses to a biotic elicitor, greenhouse-grown plant materials were inoculated with the plant pathogen, Colletotrichum gloeosporioides. Levels of hypericins increased twice as much as the control when inoculated with 1 × 10⁴ spores per ml; higher doses of spores overwhelmed the plant defenses. The elevation of hypericins and hyperforin in response to chemical and biotic elicitors suggests that these secondary metabolites are components in the inducible plant defense responses of H. perforatum.     

Biological control of cucumber powdery mildew by Tilletiopsis minor

Samenvatting Onder optimale omstandigheden konT. minor de ontwikkeling van komkommermeeldauw (Sphaerotheca fuliginea) tegengaan.Spuiten met 2×10⁷ sporen ml^–17 dagen na inoculatie met komkommermeeldauw gaf een reductie van meeldauwontwikkeling van ongeveer 90%. Wanneer een tweede bespuiting met dezelfde concentratie sporen 3 dagen na de eerste werd toegepast bleven de planten vrij van meeldauw tot ze werden opgeruimd 3 weken later.Bij een R.L. lager dan 70% en een temperatuur boven 30 °C had geen van de behandelingen succes. T. minor bleek ongevoelig voor dimethirimol (Milcurb) bij een concentratie van 125 g ml^–1, terwijl er gemakkelijk een mutant kon worden verkregen, die resistent was tegen 100 g fenarimol ml^–1, bij gelijk blijvende groeikracht en pathogeniteit ten opzichte van komkommermeeldauw, waardoorT. minor ingepast kan worden in een schema voor geïntegreerde bestrijding.     

Purification, characterization and inhibition studies of α-amylase of Rhyzopertha dominica

Rhyzopertha dominica causes extensive damage to stored wheat grains. α-Amylase, the major insect digestive enzyme, can be an attractive candidate to control the insect damage by inhibiting the enzyme through α-amylase inhibitors. R. dominica α-amylase (RDA) was purified to homogeneity by differential ammonium sulphate fractionation, Sephadex G-25 and Sephadex G-100 column chromatography. The homogenous α-amylase has a molecular weight of 52 kDa. The pH optima was 7.0 and temperature optima was 40 °C. Activation energy of RDA was 3.9 Kcal mol⁻¹. The enzyme showed high activity with starch, amylose and amylopectin whereas dextrins were the poor substrates. The purified enzyme was identified to be α-amylase on the basis of products formed from starch. The enzyme showed Km of 0.98 mg ml⁻¹ for starch as a substrate. Citric acid, oxalic acid, salicylic acid, HgCl₂, tannic acid and α-amylase inhibitors from wheat were inhibitors whereas; Ca²⁺ and Mg²⁺ were the activators of RDA. Ki values calculated from Dixon graphs with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitors were 6.9, 2.6-8.2, 3.2 mM and 0.013-0.018 μM, respectively. The Lineweaver-Burk plots with different inhibitors showed mixed type inhibition. Wheat α-amylase inhibitor showed mainly competitive inhibition with some non-competitive behaviour and other inhibitors showed mainly non-competitive inhibition with some un-competitive behaviour. Feeding trials with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitor showed significant effect of salicylic acid and oxalic acid along with wheat α-amylase inhibitor in controlling the multiplication of R. dominica.     

Role of Salicylic Acid in Systemic Resistance Induced by Pseudomonas spp. Against Pythium aphanidermatum in Cucumber Roots

Pseudomonas corrugata strain 13 and P. aureofaciens strain 63-28, applied to roots, induced systemic resistance against Pythium aphanidermatum in cucumber roots. Salicylic acid (SA) from bacterial culture or plant tissues was quantified by high performance liquid chromatography. Both strains produced SA in King's B broth and also induced cucumber root to accumulate endogenous SA one day after bacterial inoculation. Using a split root system, more SA accumulated in roots treated with bacteria than in distant roots on the opposite side of the root system in the first two days, but this difference disappeared after 3–4 days. SA levels were significantly higher in plants treated with bacteria compared to the split control, from one to five days after bacterization. SA did not inhibit mycelial growth of Pythium aphanidermatum at 100–200µgml^–1 in vitro, but higher levels inhibited mycelial growth. Zoospore germination increased at concentrations of 10–500µgml^–1, but decreased at 1000µgml^–1 compared to lower concentrations. Exogenously applied SA failed to induce local or systemic resistance against a challenge infection by the pathogen in planta. The results of this study show that exogenous applied SA does not induce systemic resistance to cucumber root rot caused by P. aphanidermatum, but endogenous SA accumulation in cucumber roots may be involved in induced systemic resistance.     

Studies on the phloroglucinol–HCl reactive material produced by squash fruit elicited with pectinase: Isolation using hydrolytic enzymes and release of p -coumaryl aldehyde by water reflux

Pathological tissues from a variety of plants turn red when treated with the histochemical reagent phloroglucinol (PG)–HCl. This induced PG–HCl reacting material has been termed both wound gum and, more recently, induced lignin or lignin-like material. We are exploring alternative approaches to better identify this induced material. In this report, we describe methods for purifying the PG–HCl reactive material from solvent-washed tissue preparations and measuring the amount of p -coumaryl aldehyde released from the purified material by boiling in water. Acorn squash fruit wall tissue was sliced, sprayed with pectinase to elicit the formation of PG–HCl reactive material and incubated up to 72 h at 27°C. Sampled tissue was washed extensively with water and organic solvents. Insoluble residues were treated with cell wall degrading enzymes to remove cell wall materials, and dimethyl sulfoxide was used to dissolve starch. Yields of residual material increased from 7.5% in the time zero samples to 29.7% after 72 h. Refluxing the purified material in water released <6 μ g p -coumaryl aldehyde g⁻¹ from preparations of tissue at time zero and 8100 μ g p -coumaryl aldehyde g⁻¹ from samples incubated for 48 h. These results suggest that the function of the material is to sequester phytoalexins, but are not consistent with the material being lignin. Published by Elsevier Science Ltd.     

Chemical control of Botrytis cinerea and Sclerotinia sclerotiorum on dwarf snap beans

Two sprays of vinclozolin (0.5 kg a.i. ha^–1) or procymidone (0.5 kg a.i. ha^–1), the first at the beginning of flowering and the second two weeks later, gave the best control ofBotrytis cinerea on dwarf snap beans (Phaseolus vulgaris). Good results were also obtained with iprodione and with thiophanate-methyl. Vinclozolin and procymidone at the same program were the most active fungicides againstSclerotinia sclerotiorum. Treatments with the latter two fungicides resulted in increased yields of pods and had no influence on colour and quality of the pods. Residue levels were below the tolerances.Samenvatting Twee bespuitingen met vinchlozolin (0,5 kg a.i. ha^–1) of procymidon (0,5 kg a.i. ha^–1), de eerste in het stadium begin bloei en de tweede 14 dagen later tijdens de peulzetting, waren voldoende omB. cinerea in stamslabonen te bestrijden. Tevens werden goede resultaten verkregen met iprodion en thiofanaat-methyl. Vinchlozolin en procymidon gaven volgens hetzelfde schema toegepast ook de beste werking tegenS. sclerotiorum. Toepassingen van deze laatste twee fungiciden leverden een hogere peulopbrengst en hadden geen invloed op de kleur en de kwaliteit van de peulen. Het residugehalte in de peulen lag beneden de toelaatbare grens.This research was subsidized by the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL).     

Toxicity of fenpropimorph to fenarimol-resistant isolates of Penicillium italicum

Fenarimol-resistant isolates ofPenicillium italicum with cross-resistance to imidazole and triazole fungicides which inhibit ergosterol biosynthesis were tested for their sensitivity to fenpropimorph. Radial growth tests on PDA showed that the isolates (n=6) lacked cross-resistance to fenpropimorph or displayed enhanced sensitivity to the fungicide (negatively correlated cross-resistance). Control of blue mold decay of oranges incited by the wild-type isolate could be achieved by dipping fruits in suspensions of fenpropimorph at a concentration of 100 mg ml^–1. Decay of oranges incited by the fenarimol-resistant isolates was controlled at the same or at a lower concentration (30 mg ml^–1), thus showing that the normal or increased sensitivity to fenpropimorph is also expressed under in vivo conditions.Samenvatting Fenarimol-resistente isolaten vanPenicillium italicum met kruisresistentie tegen imidazool-en triazoolfungiciden die de ergosterolbiosynthese remmen, werden getoetst op hun gevoeligheid voor fenpropimorf. Radiale groeiproeven op PDA toonden aan dat de isolaten (n=6) geen kruisresistentie bezaten met fenpropimorf of een verhoogde gevoeligheid voor het middel vertoonden (negatief gecorreleerde kruisresistentie). Op sinaasappels konPenicillium-rot, veroorzaakt door het wild-type bestreden worden door middel van een dompelbehandeling met fenpropimorf bij een dosering van 100 g ml^–1). Bestrijding van rot veroorzaakt door fenarimil-resistente isolaten werd verkregen bij dezelfde of een lagere dosering (30 g ml^–1); aldus werd aangetoond dat de normale of verhoogde gevoeligheid voor fenpropimorf ook in vivo tot uiting komt.     

Phytotoxicity of solanapyrones A and B produced by the chickpea pathogen Ascochyta rabiei(Pass.) Labr. and the apparent metabolism of solanapyrone A by chickpea tissues

When chickpea shoots were placed in solanapyrone A, the compound could not be recovered from the plant and symptoms developed. These consisted of loss of turgor, shrivelling and breakage of stems and flame-shaped, chlorotic zones in leaflets. In similar experiments with solanapyrone B, only 9.4% (22 μ g) of the compound taken up was recovered and stems remained turgid but their leaflets became twisted and chlorotic and some abscized.Cells isolated from leaflets of 12 chickpea cultivars differed by up to five-fold in their sensitivity to solanapyrone A and this compound was 2.6–12.6 times more toxic than solanapyrone B, depending on cultivar.Glutathione reacted with solanapyrone A in vitro reducing its toxicity in a cell assay and forming a conjugate. Measurement of reduced glutathione concentration and glutathione-S-transferase (GST) activity among cultivars showed that the differences of their means were highly significant and both were negatively and significantly correlated with their sensitivity to solanapyrone A. Treatment of shoots with solanapyrone A enhanced total, reduced and oxidized glutathione content as well as GST activity 1.26-, 1.23-, 1.50- and 1.94-fold, respectively. Similarly, treatment of shoots with the safener, dichlormid, also raised total, oxidized and reduced glutathione levels and GST activity 1.42-, 1.07-, 1.43-, 1.42-fold, respectively. Cells isolated from shoots treated with dichlormid at 150 and 300 μ g per shoot were 2.45 and 2.66 times less sensitive to solanapyrone A, with LD₅₀values of 71.5 and 77.8 μ g ml⁻¹, respectively, as compared to 29.2 μ g ml⁻¹for controls.