Infection of leaves of Arabidopsis thaliana by Botrytis cinerea: changes in ascorbic acid, free radicals and lipid peroxidation products.

Infection of leaves of Arabidopsis thaliana with conidial suspensions of the necrotrophic pathogen Botrytis cinerea resulted in a large decrease in the level of ascorbic acid and increases in intensity of a single-peak free radical and Fe(III) (g=4.27) signals in electron paramagnetic resonance (EPR) spectra. These changes were not confined to the spreading lesions or associated areas of chlorosis, but extended to other apparently healthy tissues in the infected leaves. They are, therefore, consistent with the existence of high levels of oxidative stress being generated as a result of the infection process. The expected accompanying increases in levels of the aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE), were not observed, and in the case of MDA the levels in tissue from infected plants were appreciably lower than in the healthy controls. These last findings are surprising and demonstrate a difference in the response of A. thaliana to infection with B. cinerea compared with tissues from other plant families studied previously.

Botrytis cinerea induces the formation of free radicals in fruits of Capsicum annuum at positions remote from the site of infection.

Free radical adducts of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone have been observed by electron paramagnetic resonance spectroscopy in detached fruits of Capsicum annuum investigated 5 days after infection with Botrytis cinerea. The spectra of these adducts were at a maximum within the soft rot lesion, but they could also be detected at distances up to 50 mm from the edge of the lesion in samples following main vascular bundles. At distances greater than 40 mm, the spectrum of the ascorbate radical was also seen, and at greater distances from the lesion it was the only radical detected. With samples taken from parenchyma tissue adjacent to the vascular bundles there was little adduct formation and the ascorbate radical could be detected, albeit with reduced intensity compared to healthy tissue, at distances as small as 10 mm from the edge of the lesion. This observation of chemical changes at considerable distances from the infected tissue is in contrast to previous observations on the behaviour of other markers of oxidative stress (e.g., 4-hydroxynonenal, malondialdehyde, single-peak free radical, and Fe(III) (g = 4.27) electron paramagnetic resonance signals), where their levels decreased rapidly outside of the soft rot.

Markers for oxidative stress associated with soft rots in French beans (Phaseolus vulgaris) infected by Botrytis cinerea.

The role of active oxygen species has been studied in spreading soft-rot lesions caused by the necrotrophic fungal pathogen Botrytis cinerea Pers.:Fr. in leaves of four genotypes of French bean (Phaseolus vulgaris L.). Large increases were observed for the aldehydic end-products of oxidative damage, malondialdehyde and 4-hydroxy-2-nonenal, as a result of infection in each of the genotypes studied. Similar increases were found in a stable free radical and g=4.27 Fe(III) signals, but not Mn(II) signals, in electron paramagnetic resonance spectra. These changes were accompanied by large decreases in ascorbic acid levels, with changes in the antioxidant glutathione being genotype dependent.

A potato gene encoding a WRKY-like transcription factor is induced in interactions with Erwinia carotovora subsp. atroseptica and Phytophthora infestans and is coregulated with class I endochitinase expression.

A potato gene encoding a putative WRKY protein was isolated from a cDNA library enriched by suppression subtractive hybridization for sequences upregulated 1 h postinoculation with Erwinia carotovora subsp. atroseptica. The cDNA encodes a putative polypeptide of 172 amino acids, containing a single WRKY domain with a zinc finger motif and preceded by a potential nuclear localization site. St-WRKY1 was strongly upregulated in compatible, but only weakly in incompatible, interactions with Phytophthora infestans where, in all cases, it was coregulated with class I endochitinase, associating its expression with a known defense response. Whereas St-WRKY1 was strongly induced by E. carotovora culture filtrate (CF), confirming it to be an elicitor-induced gene, no such induction was detected after treatment with salicylic acid, methyl jasmonate, ethylene, or wounding. St-WRKY1 was upregulated by treatment of potato leaves with CFs from recombinant Escherichia coli containing plasmids expressing E. carotovora pectate lyase genes pelB and pelD, suggesting that either proteins encoded by these genes, or oligogalacturonides generated by their activity, elicit a potato defense pathway associated with St-WRKY1.

A cysteine protease gene is expressed early in resistant potato interactions with Phytophthora infestans.

A potato cysteine protease (cyp) cDNA expressed at an early stage of an incompatible interaction with Phytophthora infestans was isolated. Both the nucleotide and deduced amino acid sequences are highly homologous to those of a tomato cysteine protease, CYP1. Striking protein similarity to all known cathepsins in animals, particularly cathepsin K, was also observed. However, unlike cathepsins, a granulin binding domain is located near the carboxyl terminus of the putative CYP protein. In animals, granulins bind to receptors in the plasma membrane and signal cell growth and division. A ribonuclease protection assay demonstrated that the cyp gene is tightly regulated and is induced 15 h post inoculation with P. infestans in potato leaves either with high field resistance or in which a resistance (R) gene is activated. We conclude that a common signaling pathway is activated in each form of resistance.

Bacillus subtilis BS 107 as an antagonist of potato blackleg and soft rot bacteria.

Antimicrobial substances were produced by Bacillus subtilis BS 107 in a defined medium and isolated from culture filtrate by precipitation at pH 2.5. Active fractions were extracted in ethyl acetate, acetone, and 80% ethanol and purified by thin-layer chromatography (TLC) on silica gel plates developed with an ethanol-water mixture (2:1, v/v). In each case, a band with a Rf of 0.75 formed an inhibitory zone when the TLC plates were placed in contact with agar seeded with test cultures of the Erwinia spp. The antibiotic was released into the culture medium during early stages of growth of Bacillus subtilis BS 107 but higher amounts were released in older cultures. The antibiotic was resistant to the action of nucleases, proteases, and lipase. It was stable when autoclaved twice for 35 min at 2 atm (1 atm = 101.325 kPa) in acidic, neutral, and alkaline solutions. It remained active over the pH range of 1-14 during 1 month of observation and exhibited no loss of antimicrobial activity when stored at 4 degrees C for over 1 year. Bacillus subtilis BS 107 showed activity in vitro and in vivo against Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora, the causal agents of potato blackleg and tuber soft rot. The application of an antagonist or its antibiotic to cut potato tissues prevented or reduced symptoms of the diseases. The antibiotic was active in vitro against a broad spectrum of bacterial and fungal species.

Responses of cells and protoplasts of Coffea arabica genotypes to partially purified culture filtrates produced by Colletotrichum kahawae.

Hypocotyl-derived calli of genotypes and segregating populations of Coffea arabica, differing in susceptibility to Colletotrichum kahawae, were used to produce cell suspensions and protoplasts which were exposed to partially purified culture filtrates (PPCFs) prepared from the pathogen. The growth and viability of PPCF-treated cells and protoplasts were measured using packed cell volume, fluorescein diacetate staining and a colorimetric assay involving the tetrazolium salt MTT. Differential responses of cells and protoplasts were influenced by genotype, time of exposure and PPCF concentration. Protoplasts of resistant genotypes responded differentially from more susceptible genotypes as early as 4 h after challenge with the phytotoxin, suggesting that they were more sensitive than cell suspensions to the treatments. Protoplasts exposed to PPCFs from C. kahawae may therefore be used to screen and select genotypes resistant to, or tolerant of, coffee berry disease.

Effect of Alternaria solani culture filtrate on adventitious shoot regeneration in potato.

The effect of Alternaria solani culture filtrate on adventitious shoot regeneration from tuber discs was evaluated using five potato cultivars, which were selected based on their field reaction to Alternaria solani and which represented a range of disease reactions. The culture filtrate stimulated regeneration, a response that could prove to be very useful in the wider utilization of transformation and in vitro selection technology.

Host-Pathogen Interactions : XXV. Endopolygalacturonic Acid Lyase from Erwinia carotovora Elicits Phytoalexin Accumulation by Releasing Plant Cell Wall Fragments.

Heat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defined medium containing citrus pectin as the sole carbon source. The heat-labile elicitors were highly purified by cation-exchange chromatography on a CM-Sephadex (C-50) column, followed by agarose-affinity chromatography on a Bio-Gel A-0.5m gel filtration column. The heat-labile elicitor activity co-purified with two alpha-1,4-endopolygalacturonic acid lyases (EC 4.2.2.2). Endopolygalacturonic acid lyase activity appeared to be necessary for elicitor activity because heat-inactivated enzyme preparations did not elicit phytoalexins. The purified endopolygalacturonic acid lyases elicited pterocarpan phytoalexins at microbial-inhibitory concentrations in the soybean-cotyledon bioassay when applied at a concentration of 55 nanograms per milliliter (1 x 10(-9) molar). One of these lyases released heat-stable elicitors from soybean cell walls, citrus pectin, and sodium polypectate. The heat-stable elicitor-active material solubilized from soybean cell walls by the lyase was composed of at least 90% (w/v) uronosyl residues. These results demonstrate that endopolygalacturonic acid lyase elicits phytoalexin accumulation by releasing fragments from pectic polysaccharides in plant cell walls.

Host-Pathogen Interactions : XXI. Extraction of a Heat-Labile Elicitor of Phytoalexin Accumulation from Frozen Soybean Stems.

An extract of frozen and thawed soybean (Glycine max L. Merr. cv. Wayne) stems is active, in wounded soybean cotyledons, as a heat-labile elicitor of phytoalexins. The elicitor activity of the extract is destroyed by heating to 95 degrees C for 10 minutes. The fraction that contains heat-labile elicitor activity releases heat-stable elicitor-active molecules from purified soybean cell walls. Heat-labile elicitor activity voids a Bio-Gel P-6 column and can be absorbed onto and eluted from a DEAE Sephadex ion exchange column. Using the cotyledon phytoalexin elicitor assay, maximum heatlabile elicitor activity was obtained when soybean stems were extracted with acetate buffer at pH 6.0. Addition of 1 millimolar CaCl(2) increased apparent heat-labile elicitor activity. The heat-labile elicitor stimulated maximum phytoalexin accumulation when applied to cotyledons immediately after the cotyledons were cut. Partially purified stem extracts lost heat-labile elicitor activity during storage for several days at 3 degrees C. The possible role of a heat-labile elicitor in stimulation of phytoalexin accumulation by both abiotic and biotic elicitors is discussed.