Antifungal activity of cinnamaldehyde against Fusarium sambucinum involves inhibition of ergosterol biosynthesis.

AIMS: In this study, the antifungal effect of cinnamaldehyde against Fusarium sambucinum and its underlying mechanisms were determined. METHODS AND RESULTS: Minimum inhibitory concentration and minimal fungicidal concentration of cinnamaldehyde were 3 and 4 mmol l-1 on spore germination and colony development assays in vitro, respectively. Furthermore, the lesion diameter of potato tubers and tuber slices inoculated with F. sambucinum was reduced by 76·9 and 69% after treatment with 4 mmol l-1 cinnamaldehyde. Cytometric analyses revelled that cinnamaldehyde significantly affected the integrity of cell membrane firstly, then decreased mitochondrial membrane potential and induced the accumulation of intracellular reactive oxygen species. Meanwhile, high-performance liquid chromatography results indicated that 3 mmol l-1 cinnamaldehyde could reduce the ergosterol content by 67·94%. This effect was accompanied by a down-regulation of ERG11, ERG6 and ERG4 which were involved in ergosterol biosynthesis. CONCLUSION: Theses results suggest that cinnamaldehyde exerts strong antifungal activity against F. sambucinum, probably by affecting the ergosterol biosynthetic processes what leads to the disruption of cell membrane integrity. SIGNIFICANCE AND IMPACT OF THE STUDY: Cinnamaldehyde is a predominant constituent and key flavour compound of cinnamon essential oil. It has been used as a food additive and flavorant. It is expected to be a novel and safe fungicide for controlling dry rot in potato tubes.

The dynamic changes in olive fruit phenolic metabolism and its contribution to the activation of quiescent Colletotrichum infection.

Anthracnose, the most critical disease affecting olive fruits, is caused by Colletotrichum species. While developing olive fruits are immune to the pathogen regardless of the cultivar, the resistance level varies once the fruit ripens. The defense mechanisms responsible for this difference in resistance are not well understood. To explore this, we analyzed the phenolic metabolic pathways occurring in olive fruits and their susceptibility to the pathogen during ripening in two resistant cultivars ('Empeltre' and 'Frantoio') and two susceptible cultivars ('Hojiblanca' and 'Picudo'). Overall, resistant cultivars induced the synthesis of aldehydic and demethylated forms of phenols, which highly inhibited fungal spore germination. In contrast, susceptible cultivars promoted the synthesis of hydroxytyrosol 4-O-glucoside during ripening, a compound with no antifungal effect. This study showed that the distinct phenolic profiles between resistant and susceptible cultivars play a key role in determining olive fruit resistance to Colletotrichum species.

Role of nitrogen-metabolism genes expressed during pathogenicity of the alkalinizing Colletotrichum gloeosporioides and their differential expression in acidifying pathogens.

Pathogens can actively alter fruit pH around the infection site, signaling modulation of pathogenicity-factor expression, as found for alkalinizing (Colletotrichum and Alternaria spp.) and acidifying (Penicillium, Botrytis, and Sclerotinia spp.) fungi. The nitrogen-metabolism genes GDH2, GS1, GLT, and MEP genes are differentially expressed during colonization by Colletotrichum gloeosporioides, and a Δgdh2 strain reduces ammonia accumulation and pathogenicity. We analyzed the contribution of transporters GLT and MEPB to C. gloeosporiodes pathogenicity. Germinating spores of Δglt strains showed reduced appressorium formation; those of ΔmepB mutants showed rapid ammonia uptake and accumulation inside the hyphae, indicating deregulated uptake. Both mutants reduced pathogenicity, indicating that these transporters function during alkalinizing species pathogenicity. We compared the expressions of these genes in C. gloeosporioides and Sclerotinia sclerotiorum, and found five to 10-fold higher expression at the transcript level in the former. Interestingly, GLT and MEPB in the alkalinizing species showed no and very low sequence identity, respectively, with their counterparts in the acidifying species. Knockout analysis of GLT and MEPB and their differential transcript regulation in the alkalinizing and acidifying species suggest that the ammonia accumulation contributing to pathogenicity in the former is modulated by factors at the gene-regulation levels that are lacking in the acidifying species.

Role of ammonia secretion and pH modulation on pathogenicity of Colletotrichum coccodes on tomato fruit.

Colletotrichum coccodes was found to alkalinize the decaying tissue of tomato fruit via accumulation and secretion of ammonia. Alkalinization dynamics caused by ammonia secretion from growing hyphae was examined microscopically using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Values of pH of 7.9 observed in the host tissue close to the hyphal tips declined to pH 6.0 at 10 mm away from the hyphal tip, which was a value that was still higher than that detected in the healthy tissue, pH 4.2. Ammonia accumulation at the infection site depended on the initial environmental pH. Treatments with low (4.0) pH buffer at the infection site resulted in high levels of ammonia secretion and increased virulence of C. coccodes compared with similar treatments with buffer at pH 7.0. Significantly, mutants of C. coccodes defective in nitrogen utilization, nit-, and areA- were impaired in ammonia secretion and showed reduced decay development. The reduced infection rate of nit- mutants could be complemented by adding glutamine at the infection site. Thus, ammonia accumulation is a critical factor contributing to C. coccodes pathogenicity on tomato fruit. The results show that the initial acidic pH of the fruit is conducive to ammonia secretion and the subsequent alkalinization of the infection site, and facilitates fungal virulence and the transformation from the quiescent-biotrophic to active-necrotrophic state.

Core Rot Development in Red Delicious Apples Is Affected by Susceptibility of the Seed Locule to Alternaria alternata Colonization.

ABSTRACT Alternaria alternata is the causal organism of core rot decay symptoms in susceptible cv. Red Delicious but not in resistant cv. Golden Delicious. The two cultivars did not differ in natural colonization of the style and ovary during the first week after full bloom; colonization of the ovary in the susceptible cultivar subsequently decreased with increasing distance from the calycine tube. By 30 days after full bloom, Alternaria recovery from ovary 1, adjacent to the end of the calycine tube, was 100 and 40% in the susceptible and resistant cultivars, respectively. In the susceptible cultivar, Alternaria recovery decreased from 75 to 20% in ovaries 2, 3, and 4, while there was only minor incidence in the resistant cultivar. Inoculation of the mesoderms of the two cultivars induced similar decay symptoms, but inoculated locules of Red Delicious were more susceptible than those of Golden Delicious. Increased inoculum concentration or isolate virulence enhanced the difference in locule susceptibility between the cultivars. Inoculation on isolated seed locules or on media amended with susceptible locule tissue as a carbon source induced greater transcript levels of several genes than the inoculation on resistant tissue. Endo- and exoglucanase activity levels were higher at pH 4.8 than at 4.2, conditions typical of the mesoderm adjacent to the seed locules of the susceptible and resistant cultivars, respectively. Current results suggest that susceptibility of Red Delicious apples to core rot decay is dependent on the sensitivity to locule colonization and on mesoderm pH, a factor that enhances fungal virulence.

Pathogen quiescence in postharvest diseases.

This chapter examines the quiescence period during different stages of fungal attack of postharvest pathogens: quiescence during spore germination and initial hyphal development, during and after appressorium formation, and quiescence of germinated appressorium and subcuticular hyphae. The different mechanisms for quiescence are reviewed: factors affecting quiescence of germinated spores, appressoria formation and germination, and fungal colonization. Special emphasis is given to mechanisms of quiescence involving fungal colonization: 1. the pathogen's nutritional requirements, 2. preformed antifungal compounds, 3. the elicitation of phytoalexins and preformed compounds, and 4. the activation of factors in fungal pathogenicity.

Metabolism of the Flavonoid Epicatechin by Laccase of Colletotrichum gloeosporioides and Its Effect on Pathogenicity on Avocado Fruits.

ABSTRACT During avocado fruit ripening, decreasing levels of the flavonoid epicatechin have been reported to modulate the metabolism of preformed antifungal compounds and the activation of quiescent Colletotrichum gloeosporioides infections. Epicatechin levels decreased as well when C. gloeosporioides was grown in the presence of epicatechin in culture. Extracts of laccase enzyme obtained from decayed tissue and culture media fully metabolized the epicatechin substrate within 4 and 20 h, respectively. Purified laccase protein from C. gloeosporioides showed an apparent MW of 60,000, an isoelectric point at pH 3.9, and maximal epicatechin degradation at pH 5.6. Inhibitors of fungal laccase such as EDTA and thioglycolic acid reduced C. gloeosporioides symptom development when applied to ripening susceptible fruits. Isolates of C. gloeosporioides with reduced laccase activity and no capability to metabolize epicatechin showed reduced pathogenicity on ripening fruits. On the contrary, Mexican isolates with increasing capabilities to metabolize epicatechin showed early symptoms of disease in unripe fruits. Transcript levels of cglac1, encoding C. gloeosporioides laccase, were enhanced during fungal development in the presence of epicatechin at pH 6.0, where avocado fruits are susceptible to fungal attack. But transcript increase was not detected at pH 5.0, where the fruit is resistant to fungal attack. The present results suggest that biotransformation of epicatechin by C. gloeosporioides in ripening fruits is followed by the decline of the preformed antifungal diene compound, resulting in the activation of quiescent infections.

Purification of pectate lyase produced by Colletotrichum gloeosporioides and its inhibition by epicatechin: a possible factor involved in the resistance of unripe avocado fruits to anthracnose.

Pectate lyase (PL) from Colletotrichum gloeosporioides was purified to apparent homogeneity by hydrophobic interaction chromatography followed by isoelectric focusing. The purified preparation showed one band corresponding to 40 kD on sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the enzyme was 7.9, and the optimum pH for activity was 8.9. The purified PL efficiently macerated unripe avocado fruit wedges. In vitro translation of mRNA from an induced fungal culture revealed a 36-kD precursor polypeptide, which was precipitated with PL antibodies. The antibodies inhibited enzymatic activity and maceration ability on avocado wedges. Epicatechin, a flavan 3-ol present in the peel of unripe avocado fruit, had a Ki of 3.4 microM for inhibition of PL activity in vitro. At 20 micrograms/ml (68 microM), epicatechin reduced the enzyme's macerating ability by 64%. Since the flavan is present in unripe fruit at much higher concentrations (about 350 micrograms/g fresh weight) than the inhibitory concentrations, epicatechin may be involved in the resistance of unripe avocado fruits by inhibiting the PL activity of C. gloeosporioides.

Expression of pectate lyase from Colletotrichum gloesosporioides in C. magna promotes pathogenicity.

To test the contribution of pectate lyase (PL) to promoting fungal pathogenicity, a pectate lyase gene (pel) from the avocado pathogen Colletotrichum gloeosporioides, isolate Cg-14, was expressed in C. magna isolate L-2.5, a pathogen of cucurbits that causes minor symptoms in watermelon seedlings and avocado fruits. Isolate L-2.5 was transformed with pPCPH-1 containing hph-B as a selectable marker and the 4.1-kb genomic pel clone. Southern hybridization, with the 4.1-kb genomic pel clone or 2.13-kb hph-B cassette as probes, detected integration of pel in transformed C. magna isolates Cm-PL-3 and Cm-PL-10. Western blot (immunoblot) analysis with antibodies against Cg-14 PL detected a single PL secreted by L-2.5 at a molecular mass of 41.5 kDa, whereas the PL of C. gloeosporioides had a molecular mass of 39 kDa. When PL activity was measured 4 days after inoculation in pectolytic enzyme-inducing media (PEIM), transformed isolates Cm-PL-3 and Cm-PL-10 showed additive PL activity relative to both Cg-14 and L-2.5. Transformed isolates also showed additive maceration capabilities on avocado pericarp relative to the wild-type C. magna alone, but did not reach the maceration ability of C. gloeosporioides. However, more severe maceration and damping off developed in watermelon seedlings inoculated with the transformed isolates compared with the two wild-type isolates, which showed no symptom development on these seedlings during the same period. Results clearly show the contribution of a single pel to the pathogenic abilities of C. magna and suggest that PL is a pathogenicity factor required for the penetration and colonization of Colletotrichum species.

Local modulation of host pH by Colletotrichum species as a mechanism to increase virulence.

The phytopathogenic fungus Colletotrichum gloeosporioides produces one pectate lyase (PL) that is a key virulence factor in disease development. During growth of C. gloeosporioides, Colletotrichum acutatum, and Colletotrichum coccodes in acidified yeast extract medium, the fungus secreted ammonia and increased the medium pH. Ammonia accumulation and the consequent pH change increased as a function of initial pH and buffer capacity of the medium. PL secretion by C. gloeosporioides correspondingly increased as the pH of the medium increased. The C. gloeosporioides pelB gene-disrupted mutant was able to increase ammonia accumulation and pH of the media similarly to the wild-type isolate. C. gloeosporioides in avocado, C. coccodes in tomato, and C. acutatum in apple showed ammonia accumulation in the infected area where pH increased to 7.5 to 8 and PL activity is optima. In nonhost interactions where C. gloeosporioides was inoculated in apples, the addition of ammonia-releasing compounds significantly enhanced pathogenicity to levels similar to those caused by the compatible C. acutatum-apple interaction. The results therefore suggest the importance of ammonia secretion as a virulence factor, enhancing environmental pH and pathogenicity of the Colletotrichum species.

Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction.

Colletotrichum gloeosporioides is an important pathogen of tropical and subtropical fruits. The C. gloeosporioides pelB gene was disrupted in the fungus via homologous recombination. Three independent isolates, GD-14, GD-23, and GD-29, did not produce or secrete pectate lyase B (PLB) and exhibited 25% lower pectate lyase (PL) and pectin lyase (PNL) activities and 15% higher polygalacturonase (PG) activity than the wild type. The PLB mutants exhibited no growth reduction on glucose, Na polypectate, or pectin as the sole carbon source at pH 3.8 or 6.0, except for a 15% reduction on pectin at pH 6.0. When pelB mutants were inoculated onto avocado fruits, however, a 36 to 45% reduction in estimated decay diameter was observed compared with the two controls, the wild type and undisrupted transformed isolate. In addition, these pelB mutants induced a significantly higher host phenylalanine ammonia lyase activity as well as the antifungal diene, which is indicative of higher host resistance. These results suggest that PLB is an important factor in the attack of C. gloeosporioides on avocado fruit, probably as a result of its virulence factor and role in the induction of host defense mechanisms.

Antifungal compounds from idioblast cells isolated from avocado fruits.

(E,Z,Z)-1-Acetoxy-2-hydroxy-4-oxo-heneicosa-5,12,15-triene was isolated from avocado, Persea americana Mill., idioblast cells. It inhibited spore germination of the fungal pathogen Colletotrichum gloeosporioides. Full characterization is also reported for two additional compounds that have been described and partially characterized previously.

Early Events During Quiescent Infection Development by Colletotrichum gloeosporioides in Unripe Avocado Fruits.

Inoculation of avocado pericarp tissue with Colletotrichum gloeospori-oides and treatment of avocado cell cultures with the cell wall elicitor of C. gloeosporioidesboth increased the production of reactive oxygen species (ROS). However, whereas the production of ROS could be detected within minutes in avocado cell suspensions, it was detected only after 2 h following inoculation of pericarp tissue. Protein kinase inhibitors such as K-252a and staurosporine and the phosphatase inhibitor microcystin-LR inhibited the release of H(2)O(2) from avocado cell suspensions. When 1 mM H(2)O(2) was exogenously applied to pericarp tissue, it enhanced ROS, phenyl-alanine ammonia lyase (PAL) activity, and epicatechin levels. But, when H(2)O(2) treatment was applied following staurosporine treatment, PAL activity was no longer induced. The uninduced ROS production in pericarp tissue of freshly harvested, unripe, resistant fruit was twice as high as in ripe, susceptible fruit. Challenge inoculation of resistant fruit further increased the ROS level; however, this increase did not occur in susceptible fruits. The current findings are consistent with the hypothesis that production of ROS is induced by fungal infection of unripe fruits and, consequently, may modulate resistance, resulting in the inhibition of fungal development and quiescence.

The Analysis of Fruit Protection Mechanisms Provided by Reduced-Pathogenicity Mutants of Colletotrichum gloeosporioides Obtained by Restriction Enzyme Mediated Integration.

ABSTRACT Colletotrichum gloeosporioides is an important postharvest pathogen that attacks ripe avocado fruit. Two reduced-pathogenicity mutants, Cg-M-142 and Cg-M-1150, previously obtained by restriction enzyme mediated integration, were used for the sequential analysis of the induction of biocontrol in avocado fruit. Plant biochemical indicators, such as H(+)-ATPase activity and levels of reactive oxygen species, phenylalanine ammonia lyase, epicatechin, and an antifungal diene, were investigated. The main difference between Cg-M-142 and Cg-M-1150 was the lack of appressorium formation by the latter. Preinoculation of avocado fruit with Cg-M-142 enhanced H(+)-ATPase activity and the production of reactive oxygen species. These early signaling events were followed by higher phenylalanine ammonia lyase activity and higher levels of epicatechin and the antifungal diene, and decay was delayed. Unlike Cg-M-142, Cg-M-1150 did not activate early signaling events related to fruit resistance. We suggest that the initiation of early signaling events affecting fruit resistance is determined by the capability of the pathogen to interact with the fruit during appressorium formation. Furthermore, the intensity of the fruit defense response determines the level of resistance during fruit storage.

Effect of Modified Atmosphere for Control of Black Spot, Caused by Alternaria alternata, on Stored Persimmon Fruits.

ABSTRACT Modified atmosphere packaging (MAP) of persimmon fruit resulted in the accumulation of acetaldehyde to a level of 80 mug/ml; ethanol to a level of 900 mug/ml; and CO(2) up to 30%. When fruits were stored at -1 degrees C for 4 months in such atmospheres, the incidence of black spot disease, caused by Alternaria alternata, was reduced. The effects of each of these gases were examined to determine their individual involvement in the inhibition of Alternaria development during storage. When A. alternata, grown at 20 degrees C on potato dextrose agar or inoculated in persimmon fruit, was exposed for 24 h to different levels of each volatile, acetaldehyde was the most fungistatic but only at concentrations higher than those that accumulated under MAP; CO(2) was moderately inhibitory at concentrations from 10 to 60%, whereas ethanol had no effect. Similar inhibitory effects were obtained with acetaldehyde at 620 mug/ml or 30% CO(2) when in vitro cultures of A. alternata and infected fruits were exposed for up to 2 weeks at 20 degrees C, but 1,000 mug of ethanol per ml had only a transitory inhibitory effect under these conditions. Based on analysis of the effect of concentration versus time for each gas accumulating in MAP, we suggest that the increasing concentration of CO(2) during storage is the principal factor in the inhibition of black spot disease development.

Resistance of Gibberellin-Treated Persimmon Fruit to Alternaria alternata Arises from the Reduced Ability of the Fungus to Produce Endo-1,4-beta-Glucanase.

ABSTRACT Black-spot symptoms, caused by Alternaria alternata, developed in persimmon fruits during prolonged storage at -1 degrees C. A preharvest treatment with gibberellic acid (GA(3)) extended the storage life of the fruit by delaying both black-spot development and fruit softening. Conversely, treatment of persimmon fruits with paclobutrazol (PBZ), an inhibitor of gibberellin (GA) synthesis, enhanced black-spot development and fruit softening during storage. Production of endo-1,4-beta-glucanase (EC 3.2.1.4, EG) by A. alternata in culture and in the presence of cell walls from PBZ-treated fruits as the carbon source, was enhanced by 150% over production in the presence of cell walls from control fruits, whereas endoglucanase (EG) production in the presence of cell walls from GA(3)-treated fruits was reduced by 49% relative to controls. To determine the importance of EG in symptom development, A. alternata EG was purified from a culture-inducing medium. It had a molecular mass of 41 kDa, its optimal pH and temperature for activity were 5.5 and 47 degrees C, respectively, and the pI was 4.3. Its K(m) and V(max) were 0.43 mg ml(-1) and 18 mumol reducing groups minute per milligrams of protein, respectively. The internal sequence of a 21-mer amino acid peptide from the purified EG showed 62% similarity and 38% identity to the EG-1 of Trichoderma reesei and of T. longibrachiatum. Purified EG induced black-spot symptoms on the fruit, similar to those caused by A. alternata, whereas boiled enzyme caused only pricking signs. Our results suggest that the black-spot symptoms caused by A. alternata, in persimmon, are related to the ability of the fungus to produce EG in developing lesions.

Development of Colletotrichum gloeosporioides Restriction Enzyme-Mediated Integration Mutants as Biocontrol Agents Against Anthracnose Disease in Avocado Fruits.

ABSTRACT Reduced-pathogenicity mutants of the avocado fruit pathogen Colletotrichum gloeosporioides isolate Cg-14 (teleomorph: Glomerella cingulata) were generated by insertional mutagenesis by restriction enzyme-mediated integration (REMI) transformation. Following seven transformations, 3,500 hygromycin-resistant isolates were subjected to a virulence assay by inoculation on mesocarp and pericarp of cv. Fuerte avocado fruits. Fourteen isolates showed a reduced degree of virulence relative compared with wild-type Cg-14. Two isolates, Cg-M-142 and Cg-M-1150, were further characterized. Cg-M-142 produced appressoria on avocado pericarp similar to Cg-14, but caused reduced symptom development on the fruit's pericarp and mesocarp. Isolate Cg-M-1150 did not produce appressoria; it caused much reduced maceration on the mesocarp and no symptoms on the pericarp. Southern blot analysis of Cg-M-142 and Cg-M-1150 showed REMI at different XbaI sites of the fungal genome. Pre-inoculation of avocado fruit with Cg-M-142 delayed symptom development by the wild-type isolate. Induced resistance was accompanied by an increase in the levels of preformed antifungal diene, from 760 to 1,200 mug/g fresh weight 9 days after inoculation, whereas pre-inoculation with Cg-M-1150 did not affect the level of antifungal diene, nor did it delay the appearance of decay symptoms. The results presented here show that reduced-pathogenicity isolates can be used for the biological control of anthracnose caused by C. gloeosporioides attack.

Multi-factor regulation of pectate lyase secretion by Colletotrichum gloeosporioides pathogenic on avocado fruits.

Tissue alkalinization during Colletotrichum gloeosporioides attack enhances the expression of PELB, which encodes pectate lyase (PL), and PL secretion, which is considered essential for full virulence. We studied the regulation of PL secretion by manipulation of C. gloeosporioides PELB. PELB was down-regulated by knocking out PAC1, which encodes the PacC transcription factor that regulates gene products with pH-sensitive activities. We functionally characterized a PACC gene homologue, PAC1, from C. gloeosporioides wild-type (WT) Cg-14 and two independent deletion strains, Deltapac1(372)and Deltapac1(761). Loss-of-function PAC1 mutants showed 85% reduction of PELB transcript expression, delayed PL secretion and dramatically reduced virulence, as detected in infection assays with avocado fruits. In contrast, PELB was up-regulated in the presence of carbon sources such as glucose. When glucose was used as a carbon source in the medium for the WT strain and the Deltapac1 mutant at pH 6.0, PELB transcript expression and PL secretion were activated. Other sugars, such as sucrose and fructose (but not galactose), also activated PELB expression. These results suggest that the pH-regulated response is only part of a multi-factor regulation of PELB, and that sugars are also needed to promote the transition from quiescent to active necrotrophic development by the pathogen.

Effect of ammonia production by Colletotrichum gloeosporioides on pelB activation, pectate lyase secretion, and fruit pathogenicity.

The accumulation of ammonia and associated tissue alkalinization predispose avocado fruit to attack by Colletotrichum gloeosporioides. Secretion of ammonia by C. gloeosporioides in the presence of KNO3 was induced by decreasing the pH from 7.0 to 4.0. When the fungus was grown at pH 4.0 or 6.0 in the absence of a nitrogen source, ammonia did not accumulate, and neither pelB (encoding pectate lyase) transcription nor pectate lyase secretion was detected. Under these nitrogen starvation conditions, only transcriptional activation of areA, which encodes the global nitrogen regulator, was detected. pelB transcription and pectate lyase secretion were both detected when C. gloeosporioides was grown at pH 6.0 in the presence of ammonia accumulated from different nitrogen sources. The early accumulation of ammonia induced early pelB expression and pectate lyase secretion. As the external pH increased from 4.0 to 6.0, transcripts of pac1, the C. gloeosporioides pacC homolog, also could be detected. Nit mutants of C. gloeosporioides, which cannot utilize KNO3 as a nitrogen source, did not secrete ammonia, alkalinize the medium, or secrete pectate lyase. If Nit mutants were grown at pH 6.0 in the presence of glutamate, then pectate lyase secretion was induced. Infiltration of 0.1 M ammonium hydroxide at pH 10 into ripening avocado fruits enhanced the activation of quiescent infection and symptom development by C. gloeosporioides. These results suggest that ambient pH alkalinization resulting from ammonia accumulation and the availability of ammonia as a nitrogen source independently regulate pelB expression, pectate lyase secretion, and virulence of C. gloeosporioides. These data suggest that alkalinization during C. gloeosporioides infection is important for its transformation from the quiescent biotrophic stage to the necrotrophic stage of fungal colonization in the fruit host.

Effect of particle size of activated charcoal on separation of Triton X-100 from protein, liver cytosol, and lipoxygenase extracts.

Triton X-100 was almost completely removed from bovine serum albumin solutions, BALB/c mouse liver extracts, and avocado peel lipoxygenase extracts by stirring the samples for 30 min in the presence of 250-350 mesh activated charcoal. The procedure did not remove protein significantly and did not reduce enzyme activity. At higher charcoal particle sizes, the efficiency of Triton adsorption was decreased and protein adsorption was increased. ElevateD temperatures enhanced Triton and protein adsorption. Adsorption on activated charcoal of 250-350 mesh is a simple and rapid procedure for Triton removal at a ratio of 0.23 g Triton X-100 per gram of activated charcoal.