Increased Incidence of Erwinia Soft-rot on Calla Lilies in the Presence of Phosphorous

Erwinia soft-rot is an important disease of many ornamental potted crops and is one of the most limiting factors in greenhouse calla lily (Zantedeschia spp.) production. Experiments were conducted to test the effect of phosphorous added to soil-less mixes or to nutrient solutions used for irrigation on soft-rot caused by Erwinia carotovora subsp. carotovora (Ecc). Soft-rot incidence increased to 51% when soil-less mix was amended with superphosphate in comparison to regular soil-less mix (no superphosphate added) (31%). In contrast, addition of phosphorous in the nutrient solution met the phosphorous needs of the plant without enhancing soft-rot. Plant height, fresh mass, and number of flowers per plant were greater in calla lilies irrigated with nutrient solution containing phosphorous than no phosphorous treatments. Similar results were obtained in tests conducted in a commercial greenhouse with larger sample size. No statistical differences were found between tubers sprayed with water (control) or with a 0.5 mM solution of KH₂PO₄ in laboratory experiments to determine the effect of phosphorous on tuber root development. In other experiments, tubers were sprayed with either water, a bacterial cell suspension 1 × 10² cfu ml^–1, a solution of 0.5 mM KH₂PO₄, or a suspension of bacteria in KH₂PO₄. The results from these tests showed a significant increase of soft-rot development in tubers treated with the suspension of Ecc prepared in a solution of KH₂PO₄ relative to other treatments. Further laboratory tests indicated that enzymatic activity (polygalacturonase and pectate lyase) of Ecc increased when grown in the presence of phosphorous. These experiments suggest that increased soft-rot in the presence of phosphorous is due to increased virulence of Ecc.     

Induction of defence responses against Erwinia soft rot by an endogenous pectate lyase in potatoes

Pectate lyase (PL) enzymes are major virulence factors of Erwinia carotovora (Ec) bacteria. They degrade plant cell wall pectin into unsaturated oligogalacturonates (OG) known to elicit plant defence responses. Therefore, a gene encoding the isoenzyme PL3 of Ec ssp. atroseptica was transformed by means of Agrobacterium into potatoes of cv. Désirée. Four PL-transgenic potato plant lines selected on the basis of greenhouse experiments were grown over a period of 4 years (1997–2000) in the field. It is shown that the heterologous PL enzyme mediated an enhanced resistance to Erwinia soft rot in field grown tubers. Thus compared to the non-transgenic counterpart extension of rotting was significantly reduced (P < 0.001) on the wound surface of PL-expressing tubers. Moreover, the threshold density ofEc -bacteria causing a progressive soft rot was up to 19-fold higher on tuber tissue containing the PL enzyme. An induction of plant defence responses in PL-transgenic potatoes may be indicated by an enhanced resistance of tuber tissue cell walls to Erwinia -derived enzymes, an increased PPO- and PAL-activity in tuber tissue as well as by a strengthened formation of necrosis on the wound surface of tubers after infection with Ec -bacteria.     

Digalacturonates from pectin degradation induce tissue responses against potato soft rot

The pathogenicity of the bacteriumErwinia carotovorasubsp.atroseptica, which causes potato soft rot, is triggered by short oligogalacturonates released by enzymic degradation of plant cell wall pectin. In the first stage unsaturated digalacturonate (uDG), produced by the action of pectate lyases, is degraded by oligogalacturonide lyase (OGL) to keto-deoxyuronate (DKI). The OGL encoding gene fromE. carotovoraand the corresponding recombinant enzyme were characterized. Measuring the changes in plant cell viability and tissue maceration during soft rot pathogenesis in tissue slices of sprouting potato tubers, it was observed that exposure to uDG and DKI, produced by recombinant OGL, killed up to 30% of the plant cells over a period of 16h. This protected the tissue against maceration byE. carotovorasubsp.atroseptica. Endogenous OGL activity was detected in extracts from sprouting tubers where it may be involved in the conversion of uDG into cell toxic compounds. The results indicate that an additional function of small, diffusable digalacturonates is to induce plant cell death during the rotting process, thus contributing to defence reactions againstE. carotovorasubsp.atroseptica.     

A rapid method to identify and quantify soft rot erwinias on seed potato tubers1

Potato tubers are usually contaminated by more than one species or pathovar of soft rot erwinia and, because blackleg incidence is related to the contamination level of seed tubers, the disease potential of seed stocks may be assessed by determining seed-tuber contamination level. A method is described for identifying and quantifying directly from tubers the three soft rot erwinias commonly associated with potatoes. Replicate lots of 10–15 tubers are peeled by dry abrasion in a commercial potato peeler and the peel weight determined by weighing the tubers before and after peeling. Sap is expressed from the peel, an antioxidant (0.075% dithiothreitol) added, and the sap is dilution-plated on a diagnostic selective medium (crystal violet pectate [CVP]). After incubating for 24 h at 20°C, the plates are velvet-replicated onto fresh plates of CVP with or without 35 μg ml^-1 erythromycin and incubated for 48 h at 27°C and 24 h at 33.5 or 37°C. Soft-rot erwinias typically form deep cup-like cavities on CVP and they can be identified and enumerated according to the pattern of cavity formation. Cavities are formed by Erwinia carotovora pv. atroseptica only at 27°C, by E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C, whereas E. chrysanthemi forms cavities at all temperatures but fails to grow in the presence of erythromycin. Contamination levels can be expressed as the number of different erwinias per tuber or per g peel.     

Moss-Erwinia pathosystem reveals possible similarities in pathogenesis and pathogen defense in vascular and nonvascular plants

Vascular plants have various inducible resistance mechanisms as defense against pathogens. Mosses, small nonvascular plants (subkingdom Bryophyta), have been little studied in regard to their pathogens or modes of defense. Data here show that Erwinia carotovora, a bacterial plant pathogen that causes softrot in many dicotyledonous plants, can also cause soft rot symptoms in the moss Physcomitrella patens. Infection of moss by E. carotovora required pathogenicity factors similar to those required to infect vascular plants and, again as in vascular plants, salicylic acid (SA) induced moss to inhibit tissue maceration by Erwinia. These data reveal that SA-dependent defense pathways may have evolved before differentiation of vascular and nonvascular plants.     

Reaction of saprophytic bacteria from potato peel extracts and plant pathogenic bacteria in ELISA with antisera to Erwinia chrysanthemi (serogroup O1Ha)

The specificity of two antisera raised to whole cells ofErwinia chrysanthemi (Ech), serogroup O₁H_a, was studied in double antibody sandwich (DAS-) ELISA with 100 strains of different plant pathogenic bacteria (PPB), including 39 Ech strains, and of one of these antisera with 900 saprophytic bacteria isolated from extracts of potato peelings of Dutch seed potatoes grown in several production areas.All tested European Ech strains from potato reacted positively while no reactions were observed with any of the other plant pathogenic bacterial species. Two saprophytes (A254 and A256), both identified as pectinolyticPseudomonas fluorescens species, cross-reacted strongly with polyclonal antibodies against Ech. Non-specific reactions were found in DAS-ELISA with 16 saprophytes. The detection limits for the individual saprophytes varied between c. 10⁵ and 10⁹ cells.ml^–1. The non-specific reactions were also found with monoclonal antibodies (mca 2A4) against a proteinase K resistent epitope of Ech and with antisera against other plant pathogens including an antiserum against potato virus Y^N. The non-specific reactions were observed in DAS-ELISA, but not in Ouchterlony double diffusion or immunofluorescence colonystaining, whereas A254 and A256 reacted in all tests, but only with antibodies against Ech. When in making dilution series potato peel extracts were used instead of phosphate buffered saline with 0.1% Tween 20, the 14 non-specifically reacting saprophytes only reacted at concentrations of 10⁹ cells.ml^–1 or higher. Only one of these 14 saprophytes was able to multiply on injured potato tuber tissue.In contrast to most saprophytic strains, the saprophytes A254 and A256 reacted strongly in ELISA in dilutions series made with potato peel extracts. A256 was able to grow on potato tuber tissue but only under low oxygen conditions; A254 did not grow at all on potato tissue.Defatted milk powder or bovine serum albumin added to the dilution buffer for the enzymeconjugated antibodies, drastically reduced the non-specific reactions, but not the reactions with A254 and A256.To reduce the cross-reaction with A254, an Ech antiserum was absorbed with A254. This resulted in a substantial drop in antibody reaction with the homologous antigen in Ouchterlony double diffusion.     

Interpretation of the EC method for the detection of latent Corynebacterium sepedonicum infections in potato1

The EC method for the detection of latent ring-rot infections (Corynebacterium sepedonicum) consists essentially of an indirect immunofluorescence test and a pathogenicity test on eggplant (EP). When interpreting the results of this method, care should be taken that: (a) eggplants are grown at 21°C. At 28–29°C the detection level of the EP was increased 10² to 10³-fold to 10⁵ to 10⁶ cells ml^-1 and latent infections (which may go unnoticed) occurred with 10⁴ to 10⁵ cells ml^-1; (b) reisolations are made from symptomless eggplants grown at the optimum temperature (21°C) as latent infections can also occur in these plants at concentrations of 10² to 10³ cells ml^-1; (c) potato tuber extracts are tested without freezing or stored freeze-dried or in a proper protective agent. Freezing at -20°C in maceration buffer of cells of C. sepedonicum and Pseudomonas solanacearum (the latter was included for comparison) reduced their numbers by 90–98% after one day. This could easily cause viable cell numbers to drop below the detection level of the pathogenicity test.     

The role of the seed tuber in determining partial resistance to potato blackleg caused byErwinia spp.

In 1991 and 1992, 12 potato cultivars were screened at two locations for resistance to blackleg, after vacuum infiltration of the seed withErwinia carotovora subsp.atroseptica orE. chrysanthemi. Cultivar differences for resistance toE.c. subsp.Atroseptica andE. chrysanthemi were found which were consistent over locations and years. Seed tubers of the same cultivars were also screened for resistance to bothErwinia spp. by using a tuber slice inoculation method. Correlation coefficients for comparisons between resistance to blackleg in the field and tuber tissue resistance under aerobic or anaerobic conditions were not significant. This could partly be explained by drastic changes in relative tuber tissue resistance of the cultivars within a 5 weeks period after planting in the field. Presprouting of seed tubers in diffuse daylight had a less pronounced effect on relative tuber tissue resistance than planting in the field. Monitoring the process of mother tuber decay during the growing season of 1993 after vacuum infiltration withE.c. subsp.atroseptica andE. chrysanthemi revealed that cultivars differed in the extent to which these bacteria enhanced the process of mother tuber decay. These differences partly explained the cultivar differences for resistance to blackleg in the field.Abbreviations Eca Erwinia carotovora subsp.atroseptica - Ech Erwinia chrysanthemi - NOP Noordoostpolder - Wag Wageningen     

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.     

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.     

Bacterial diseases of potatoes in the major potato-growing areas in Turkey1

In 1988 and 1989 bacterial diseases of potatoes in Central Anatolia were investigated by utilizing the EC method for the detection of ring-rot bacterium (Clavibacter michiganensis ssp. sepedonicus) and using selective medium. 91 samples containing 200 tubers from commercial and farmers’ stores in Central Anatolia were tested for the presence of C.m.sepedonicus and about 50 tubers of each sample were also tested for the presence of soft-rot erwinias. Five different bacteria that cross-reacted with C.m.spedonicus antisera were isolated from heel-end extracts. All cross-reacted bacteria differed from C.m.sepedonicus morphologically and none of them was pathogenic on eggplant. C.m. sepedonicus was not detected, but Erwinia carotouora ssp. carotouora and Erwinia carotovora ssp. atroseptica were present in 7 and 17% of the samples respectively.     

Detection of the phytotoxin coronatine by ELISA and localization in infected plant tissue

Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR)in vitro, and the availability of purified toxin has facilitated development of immunological detection methods. A modified, indirect competitive ELISA using the COR-specific monoclonal antibody 11B8 was developed to detect COR in various host plants infected by P. syringae. The estimated detection limit for COR was 50 pg per well, and COR could be reliably quantified from 5 to 40 ng ml⁻¹. The subcellular localization of COR within infected tomato tissue was investigated using the COR-specific antibody MAb 8H3G2. Immunofluorescence microscopy and immunogold labelling showed that COR was present inside tomato cells and was associated with chloroplasts and particles of proteinase inhibitor I. Localization studies indicated that COR is mobile in infected plant tissue and can be detected in healthy tissue adjacent to the bacterial lesions.     

Polygalacturonase isoenzymes and oxalic acid produced by Sclerotinia sclerotiorum in soybean hypocotyls as elicitors of glyceollin

The polygalacturonases (PG) and oxalic acid produced by Sclerotinia sclerotiorum in infected soybean hypocotyls were investigated as elicitors of the phytoalexin glyceollin I.Purification to homogeneity through isoelectrofocusing and ion-exchange fast protein liquid chromatography revealed three endo-PG isoenzymes (PG-I, PG-II and PG-IV) and one exo-PG (PG-III) in 6-day-old etiolated soybean hypocotyls infected with the B-24 isolate of S. sclerotiorum.PG-I and PG-III, in the range of concentrations tested (0·15–1·2 reducing units ml⁻¹), did not act as elicitors of glyceollin I synthesis. Some elicitor activity was shown by PG-II at 0·6–1·2 reducing units ml⁻¹. PG-IV, at lower doses (0·038–0·30 reducing units ml⁻¹), was even more effective in inducing phytoalexin synthesis. However higher concentrations of PG-IV induced tissue softening and decreased phytoalexin accumulation.PG-II and PG-IV released heat-stable elicitors from purified soybean cell walls supporting the evidence that uronides are intermediate inducers in elicitation by endo-PGs. Oxalic acid was an active elicitor of glyceollin I over the range of concentrations tested (0·31–20 m ) with the maximum at a concentration of 5 m . The inability of oxalic acid to release uronides from purified cell walls makes it unlikely that uronide intermediate elicitors are involved in elicitation by oxalic acid.     

Comparison of some molecular, enzymatic and antifungal properties of chitinases from thorn-apple, tobacco and wheat

Chitinases (E.C.3.2.1.14) were isolated from leaves of thorn-apple (Datura stramonium), tobacco (Nicotiana tabacum) and from embryos of wheat (Triticum aestivum). All three chitinases exhibited similar physico-chemical, enzymatic and antifungal properties. However, the chitinase from wheat differed slightly from the other two in that it did not yield a line of identity upon immunodiffusion against Datura stramonium chitinase antiserum, and did not produce the same oligosaccharide pattern when used to hydrolyse chitin. All three chitinases inhibited germination of Trichoderma hamatum and Phycomyces blakesleeanus spores at concentrations as low as 8 and 32 μg ml⁻¹ respectively. Hyphal growth of T. hamatum and P. blakesleeanus was inhibited by 50%, at chitinase concentrations of 2 and 20 μg ml⁻¹ respectively. However, at this concentration and indeed at concentrations as high as 320 μg ml⁻¹, spore germination and hyphal growth of Botrytis cinerea was not affected by the chitinases.     

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.     

Potato plant response to seed tuber bacterization in the field in various rotations

The effects of a seed tuber treatment with antagonistic isolates of fluorescentPseudomonas spp. were investigated on potato plants from 1981 to 1984. The experimental plots were located in fields in short and long rotations of potato. The short rotations are characterized by serious yield reductions which are caused by unknown microbial factors. The reductions varied from 30% in 1982 to only 3% in 1983 in the 3-year rotations. A statistically significant increase in yield (four to five months after planting) of ware potatoes varying from 9 to 20% was obtained in these plots through tuber bacterization, but only in 1981. In 1982 and 1983 initially significant improvements in shoot or tuber weight of seed potatoes were no longer detectable at ware potato harvest at the end of the growing period. Seed tuber bacterization had no effect on tuber yield in long rotations. Initial colonization of basal root parts by 53×10⁴ colony forming units (cfu) of antibiotic-resistant mutants per gram of root (fresh) dropped significantly to 20×10⁴ cfu per gram after three months. The bacterization effect on tuber yield depended on the development of harmful microbial activity and of introduced antagonists during the growing period. Seed tuber bacterization is more promising for seed potatoes than for ware potatoes in short rotations, the latter being harvested two months later.Samenvatting De invloed van pootgoedbehandeling met antagonistische isolaten van fluorescerendePseudomonas-soorten op de aardappelteelt, werd onderzocht in de periode van 1981 tot en met 1984. De proefvelden maakten deel uit van zowel ruime als nauwe rotaties met aardappelen. Kenmerkend voor de nauwe rotatie is, dat de opbrengst aanzienllijk gereduceerd wordt als gevolg van de aanwezigheid van nog onbekende microbiële factoren. Deze opbrengstverlaging varieerde van 30% in 1982 tot slechts 3% in 1983 in de 3-jarige rotaties. Pootgoedbacterisatie had in deze proefvelden een significante toename van de eindopbrengst (vier tot vijf maanden na pootdatum) van consumptieaardappelen tot gevolg, die varieerde van 9 tot 20%, echter allen in 1981. In 1982 en 1983 werd het effect van bacterisatie ook in de loop van de groeiperiode onderzocht. Aanvankelijk significante toenames van zowel spruit-als knolgewicht waren aan het einde van het groeiseizoen niet meer aantoonbaar. Pootgoedbacteristie bleek geen effect te hebben op aardappel in ruimte rotaties. Aanvankelijk werden de basale wortelgedeelten gekoloniseerd door antibioticum-resistente mutanten met 53×10⁴ kolonievormende eenheden (kve) per gram wortel(vers); dit aantal liep (drie maanden na pootdatum) echter significant terug tot 20×10⁴ kve per gram. Het effect van bacterisatie op de eindopbrengst werd bepaald door de ontwikkeling van de schadelijke microbiële activiteit en de ontwikkeling van de geïntroduceerde antagonisten tijdens het groeiseizoen. Pootgoedbacterisatie in nauwe rotaties biedt meer mogelijkheden voor de teelt van pootaardappelen dan die van consumptieaardappelen, die geruime tijd later geoogst worden.     

Molecular cloning of an endo-pectate lyase gene from Erwinia carotovora subsp. atroseptica

We report the construction of a clone library of the Erwinia carotovora subsp. atroseptica genome and the isolation of a gene for endo-pectate lyase. The library, inserted into the PstI site of plasmid pBR322, contains approximately 1700 clones. Five of these produce pectolytic enzymes as detected by a plate screening assay. Using a cloned endo-pectate lyase gene from the related bacterium, E. carotovora subsp. carotovora, as a probe, we found that one pectolytic E. carotovora subsp. atroseptica clone had strong homology to the probe. We characterized that clone by restriction endonuclease mapping and studied its pectolytic protein product. The purified enzyme is an endo-pectate lyase with a cofactor preference for Co²⁺. The molecular weight of the protein is 31 000 and it has an isoelectric focusing point of 9·2, corresponding to an endo-pectate lyase produced by E. carotovora subsp. atroseptica, but not to the protein product of the E. carotovora subsp. carotovora probe DNA, which has a pI of 9·5. Restriction endonuclease site polymorphisms in the two cloned endo-pectate lyase genes suggest substantial sequence divergence between these two loci.     

Characterization of an antibacterial substance produced by <Emphasis Type="Italic">Erwinia carotovora</Emphasis> subsp. <Emphasis Type="Italic">carotovora</Emphasis> Ecc 32

A total of 88 strains of Erwinia carotovora subsp. carotovora (Ecc) isolated from various host plants in several geographic regions were screened for production of antibacterial substances using the same strains as indicators. Of the 88 strains, 72 produced antibacterial substances. One of these 72 strains, a Brazilian strain Ecc 32, produced an antibacterial substance active against all tested Ecc strains on TSA medium. The antibacterial spectrum of the compound from Ecc 32 strain was limited to closely related strains of soft-rot Erwinia species. Such a narrow spectrum of activity is typical of bacteriocins. The compound produced by Ecc 32 strain, however, was resistant to some enzymes and detergents. Moreover, the compound was heat-stable and active over a wide pH range. The physical characteristics of the compound were not in agreement with those of bacteriocin or carotovoricin.     

Physiological changes in Nicotiana tabacum leaves during development of chlorosis caused by coronatine

Coronatine is a non-specific, chlorosis-inducing phytotoxin produced by several pathovars of Pseudomonas syringae, including pv. atropurpurea, the causal agent of bacterial blight of ryegrass. The physiological development of chlorosis caused by coronatine was investigated in Nicotiana tabacum, a plant which was found to be particularly sensitive to this toxin. The disappearance of chlorophylls from the affected tissue was independent of light intensity, indicating that chlorosis was not the consequence of photochemical bleaching. The loss of chlorophylls was associated with a proportional decline in the rate of photosynthesis, expressed as nmol O₂.cm⁻² min⁻¹, and protein synthesis, expressed as pmol leucine incorporated cm⁻² min⁻¹. However, expressed on a chlorophyll basis, neither photosynthesis nor protein synthesis were reduced in the coronatineaffected tissue, compared to controls, revealing the physiological competence of the chlorotic tissue. Quantitative changes were observed in the tissue content of free amino acids. During the development of chlorosis there was an increase in the tissue content of tryptophan and asparagine, while the content of alanine and aspartate declined. Coronatine-affected leaves were visibly thickened, and this was associated with an increase in the number of cells in the palisade mesophyll layer. This latter observation indicates that coronatine may interfere with the growth regulation of tobacco leaves.     

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.