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Greenhouse experiments were conducted to determine disease progression of phytophthora root rot on nonwounded and wounded pepper plants (Capsicum annuum) and to determine whether susceptibility to Phytophthora capsici decreases with wound aging. Two isolates of P. capsici were used in this study, one less aggressive than the other. Trimming the roots immediately prior to inoculation with either isolate increased susceptibility significantly (P ≤ 0·05) compared with plant roots that were not trimmed. Both isolates caused a higher level of disease severity on disturbed/trimmed than on disturbed/nontrimmed roots. Disease also occurred earlier with the more aggressive isolate on both wounded and nonwounded roots. Disease severity was three to four times more severe on plants treated with the aggressive isolate (NM6011) than on those inoculated with the less aggressive isolate (NM6040), regardless of root treatment. In separate experiments, pepper roots were wounded and allowed to age for up to 5 days before inoculation. Resistance to P. capsici increased as the wounds aged, resulting in significantly (P ≤ 0·001) lower disease severity on plants with 3- and 5-day-old wounds than on those inoculated at the time of wounding and the nondisturbed/nontrimmed controls. Wounding of the roots followed by immediate inoculation with zoospores resulted in significantly higher levels of attachment than when roots were inoculated with zoospores 48 h after wounding. The 48-h postwounding inoculation treatment showed the same amount of zoospore attachment as nonwounded roots. Increase in plant resistance correlated (P ≤ 0·01) with an increase in total peroxidase activity. Isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) indicated increased band intensity of three acidic and one basic isozyme as wounds aged. These data suggest that wound repair plays a role in decreasing infection and resultant disease symptoms of pepper to P. capsici.

Fusarium solani is one of the more important fungal pathogens involved in pre- and post-harvest decay of muskmelon fruit. Production of polygalacturonase (PG), by F. solani was studied in vitro and in vivo . The fungus produced at least 14 PG isozymes with pIs of 4.5 to 9.5 in shake culture using pectin as the sole carbon source. When glucose and pectin were used in combination as the carbon source, total PG activity decreased substantially as compared to pectin alone, suggesting that glucose may suppress PG production in vitro . Only one PG isozyme, designated as PG1, was detected in extracts from infected fruit tissue. PG1 from decayed fruit was purified to homogeneity by protein extraction, ultrafiltration, gel filtration chromatography, and cation exchange chromatography. The molecular weight of PG1 was estimated at 38 kDa based on SDS-PAGE with a pI of 9.5 according to IEF-PAGE. PG1 exhibited only endo-PG activity based on viscosity reduction and thin layer chromatography analysis of products released by enzymatic action. The optimum pH for PG1 activity was 6. The K m and V max of PG1 using polygalacturonic acid as the substrate were 1.34 mg ml -1 and 0.30 unit μg protein -1 , respectively. PG1 effectively macerated fruit tissue which suggests that it may play an important role in decay of muskmelon fruit caused by F. solani .

Phomopsis cucurbitae is a latent infecting pathogen that infects unripe muskmelon fruit, but causes decay after harvest. This fungus causes severe losses during muskmelon fruit storage and marketing in the U.S., Japan, and some Central American countries. Previous studies showed that the fungus produced the cell wall-degrading enzyme polygalacturonase (PG) in both culture and muskmelon fruit tissue. The role of P. cucurbitae PG in the fruit decay process and its relation to latent infection is not well-understood. A prominent PG isozyme produced by the fungus in decayed fruit was purified to homogeneity by a sequence of extraction, ultrafiltration, preparative isoelectric focusing, anion exchange, and gel filtration chromatography. This isozyme exhibited endo-activity, a molecular weight of 54 kDa according to SDS-PAGE, and a pI of 4.2 based on IEF-PAGE. Isozyme activity was optimal at 40–45°C and pH 5.0. It had a Km of 44.7 g/ml and a Vmax of 0.313. The purified isozyme also effectively macerated mature muskmelon fruit tissues. This isozyme was the most prominent of the PG isozymes produced by P. cucurbitae in decaying fruit, and may play an important role in postharvest decay.

An isolate (87) of the yeast Pichia guilliermondii, protects apples from postharvest fruit rotting fungi Botrytis cinerea and Penicillium expansum. In order to examine the yeast-pathogen interaction, B. cinerea was grown on agar plates overlayed with cellophane. Effective and non-effective yeast isolates were applied near the young hyphal growth. Samples were taken 24 h later from the area where the fungi and yeast had intersected. Light microscopy revealed a general attachment of the effective biocontrol agent P. guilliermondii (isolate 87) and a non-effective isolate (117) of Debaryomyces hansenii. Low temperature scanning electron microscopy (LTSEM) indicated that both species of yeast attached to the fungal hyphae, but the 87 isolate attached fastidiously. Twenty-four hours after applying the 87 isolate to B. cinerea, pitting and collapse of the hyphae were observed. These observations were confirmed using transmission electron microscopy. These features were not observed with the ineffective isolate of D. hansenii. Further experiments indicated that attachment of P. guilliermondii to hyphae of B. cinerea could be blocked by agents that alter protein integrity (salts, proteases, etc.) and certain sugars. Isolates of both species produced β-(1–3) glucanase when cultured in various carbon sources and on cell walls of fruit rotting pathogens. Culture supernatants from P. guilliermondii, however, yielded two- to five-fold more β-(1–3) glucanase activity compared with D. hansenii. Data indicate that tenacious attachment, along with secretion of cell wall degrading enzymes, may play a role in the biocontrol activity of this yeast antagonist.

Xylem sap from apple (Malus domestica Borkh), peach (Prunus persica Batsch), and pear (Pyrus communis L.) twigs was collected by means of pressure extrusion. This sap contained a number of acidic peroxidases and other proteins. Two other sources of xylem sap used in this study were stem exudates and guttation fluid. Similar peroxidases were also found in stem exudates and guttation fluids of strawberry (Fragaria x ananassa Duch.), tomato (Lycopersicum esculentum L.), and cucumber (Cucumis sativus L.). Isoelectric focusing activity gels showed that two peroxidases (isoelectric point [pl] 9 and pl 4.6) were present in initial stem exudates collected in the first 30 minutes after excision. Subsequent samples of stem exudate collected contained only the pl 4.6 isozyme. The pl 4.6 peroxidase isozyme was also found in root tissue and guttation fluid. These observations suggest that roots produce and secrete the pl 4.6 peroxidase into xylem sap. Cucumber seedlings were treated with 100 microliters per liter ethylene for 16 hours and the exudate from decapitated hypocotyl stumps was collected over a 3 hour period. Ethylene increased the peroxidase activity of stem exudates and inhibited the amount of exudate released. These observations suggest that xylem sap peroxidase may play a role in plugging damaged vascular tissue.

Developing peach ( Prunus persica L. Batsch `Redskin9) fruit were used to characterize the role of peroxidases in lignification. During development, the endocarp of these drupes becomes lignified while the mesocarp remains parenchymatous. Acidic peroxidase from lignifying endocarp were similar to those of the fleshy mesocarp. The endocarp had a larger amount and number of basic peroxidases than the mesocarp. Cultured peach leaf cells are thought to be lignified because their walls give a positive reaction with phloroglucinol-HCI. These cells also secreted a basic peroxidase. Peroxidases were difficult to extract from endocarp tissue as they lignified. This was also demonstrated by tissue printing on nitrocellulose. Flesh, but not endocarp peroxidase was evident in tissue prints. This suggests that tissue printing may fail to reveal the presence of enzymes which are firmly attached to the cell.

Production of polygalacturonase (PG), a cell wall-degrading enzyme, by Phomopsis cucurbitae (latent infection fungus) was studied in relation to different carbon sources and various stages of cantaloupe fruit development. P. cucurbitae produced multiple PG isozymes both in vitro and in vivo. The fungus produced the highest PG activity and the greatest number of isozymes on pectin compared with those produced on glucose, galactose, and sucrose. Eight P. cucurbitae PG isozymes (pIs 3.7, 4.2, 6.6, 7.0, 7.3, 7.5, 7.8, and 8.6) were detected in extract from inoculated mature fruit (40 days after anthesis) by isoelectric focusing. Isozyme bands with pIs of 4.2, 7.3, and 7.8 were the most prominent. A similar set of PG isozymes was produced by P. cucurbitae in autoclaved mature fruit tissue (mesocarp). When tissue discs taken from 20-, 30-, 40-, and 50-day postanthesis fruit were inoculated with P. cucurbitae, PG activity and the number of PG isozymes extracted from the macerated fruit tissue discs increased with the degree of fruit maturity and ripening. Increases in PG activity and PG isozymes were also correlated with reactivation of latent infections and the beginning of tissue maceration. An anionic PG isozyme (pI 4.2) was only visualized on decayed 50-day-old fruit exocarp, as well as 40- and 50-day-old fruit mesocarp. The experimental results support the hypotheses that P. cucurbitae PG isozymes play an important role in fruit decay once latent infection becomes active following harvest.

Experiments were conducted to determine the relationship between pepper fruit susceptibility to Phytophthora fruit rot and fruit maturity. Pepper fruits were harvested weekly throughout the 1991 growing season and were dipped in a suspension of Phytophthora capsici zoospores (5×10 3 zoosporesl/ml). The susceptibility of nonwounded fruit to infection decreased with increased ripening. Lesion elongation in nonwounded fruit decreased from 14.1 mml/day in green fruit to 10.7 mm/day in nondehydrated red fruit. Nonwounded fruit (immature green, mature green, and red) inoculated with 5×10 3 zoospores in a 100-μl drop also exhibited less infection as the fruit ripened [...]

Ethylene enhanced the levels of peroxidases in the roots, stems, leaves, and cotyledons of 2-week-old cucumber Cucumis sativus cv Poinsett 76 seedlings. Antibodies to the isoelectric point (pl) 9 and pl 4 isoenzymes were used in a radial immuno-diffusion assay to demonstrate that ethylene induced similar peroxidases in other cultivars of C. sativus, other species of Cucumis and other genera of Cucurbitaceae. Examination of ethylene-induced peroxidases, using isoelectric focusing gels, demonstrated the presence of a series of other peroxidases, mostly slightly acidic, whose isoelectric focusing pH was approximately 6. These pl 6 peroxidases were partially purified on a cation exchange column. Ouchterlony double diffusion gels indicated that these proteins cross-reacted with antibodies to both the pl 9 and pl 4 peroxidase. The data presented here suggest that the induction of peroxidase isoenzymes during ethylene-induced senescence is a common response in this family of plants. In addition, antibody and isoelectric focusing studies indicate that both acidic and basic peroxidase are highly conserved in members of this family.