Penicillium digitatum Suppresses Production of Hydrogen Peroxide in Host Tissue During Infection of Citrus Fruit

ABSTRACT During the infection of citrus fruit by Penicillium digitatum there is little evidence of a host defense response. This suggests that P. digitatum has the ability to suppress host defenses. The current study demonstrates that P. digitatum suppresses a defense-related hydrogen peroxide (H(2)O(2)) burst in host tissue. In contrast, the nonhost pathogen, Penicillium expansum, triggers production of a significant amount of H(2)O(2) in citrus fruit exocarp. Using laser scanning confocal microscopy, we demonstrated that P. digitatum suppressed an elevation in H(2)O(2) up to 42 h after inoculation. Nevertheless, H(2)O(2) levels around wounds inoculated with P. expansum increased by 63-fold above the control. P. digitatum continued to suppress H(2)O(2) production in citrus fruit exocarp up to 66 h postinoculation and H(2)O(2) levels were actually threefold below that of noninoculated controls. In contrast, the H(2)O(2) level was still about 11-fold above the control value in wound sites inoculated with P. expansum. Studies on the effect of organic acids (as pH modulators) on the response of citrus fruit to compatible and noncompatible pathogens indicated that pathogenicity was enhanced only when host-tissue acidification was accompanied by the suppression of H(2)O(2). Additionally, pathogenicity of both P. digitatum and P. expansum on citrus fruit was significantly enhanced by the H(2)O(2)-scavenging enzyme catalase. Based on our study and previous reports regarding the potential involvement of citric acid and catalase in green mold pathogenesis, we suggest that these compounds are strongly associated with the virulence of P. digitatum.     

Prestorage heat treatment reduces pathogenicity of Penicillium expansum in apple fruit

Penicillium expansum is one of the main postharvest pathogens of apples in Israel. Heating apple fruit inoculated with P. expansum for 96 h at 38°C completely inhibited decay development. Fruit held for 24 h at 42°C or 12 h at 46°C had significantly reduced decay after an additional 14 days incubation at 20°C, compared with unheated inoculated control fruit. Mycelial growth and percentage spore germination in vitro were inversely proportional to length of time of exposure to various temperatures. The ET₅₀ for spore germination was 42, 34 and 20 h at 38, 42 and 46°C, respectively, while the ET₅₀ for mycelial growth was 48, 44 and 36 h at those temperatures. When Penicillium spores were incubated on crude extract prepared from the peel of apple fruits held 4 days at 38°C, germ tube elongation was significantly reduced, while the walls of the tubes were thicker, compared with germ tubes from spores incubated on crude extract prepared from peel of non-heated fruit. The evidence presented here supports the hypothesis that the effect of heating on the decay of apples caused by P. expansum is not only the result of direct inhibition of fungal germination and growth by high temperature, but is also partly due to the formation of an inhibitory substance in the heated peel.     

Enhancement of Biocontrol Activity of Cryptococcus laurentii by Silicon and the Possible Mechanisms Involved

ABSTRACT Exogenous application of silicon (Si) in the form of sodium metasilicate reduced disease development caused by Penicillium expansum and Monilinia fructicola in sweet cherry fruit at 20 degrees C. The inhibition of fruit decay was correlated closely with Si concentrations. Silicon at concentrations of 1%, in combination with the biocontrol agent Cryptococcus laurentii at 1 x 10(7) cells per ml, provided synergistic effects against both diseases. Population dynamics of C. laurentii were stimulated by Si 48 h after the yeast treatment in the wounds of sweet cherry fruit. Silicon strongly inhibited spore germination and germ tube elongation of P. expansum and M. fructicola in vitro. Based on results with scanning electron microscopy, growth of both pathogens was significantly inhibited by Si in the wounds of sweet cherry fruit. Compared with the wounded water control, Si treatment induced a significant increase in the activities of phenylalanine ammonia-lyase, polyphenoloxidase, and peroxidase in sweet cherry fruit but did not increase the levels of lignin. Application of Si activated a cytochemical reaction and caused tissue browning near the site of wounding. Based on our studies, the improvement in biocontrol efficacy of antagonistic yeast when combined with Si may be associated with the increased population density of antagonistic yeast by Si, the direct fungitoxicity property of Si to the pathogens, and the elicitation of biochemical defense responses in fruit.     

柑橘绿霉病菌中多聚半乳糖醛酸酶基因(PdPG2)的表达分析

柑橘绿霉病菌Penicillium digitatum是储藏期柑橘腐烂病最主要的病原之一,严重影响柑橘产业的发展。已有研究表明,柑橘绿霉病菌中多聚半乳糖醛酸酶(PdPG2)对其致病性有重要作用,PdPG2基因功能缺失突变株的致病性会下降,然而有关PdPG2基因的表达研究尚不完善。本文研究了PdPG2基因在不同条件下的表达情况,结果表明PdPG2是酸性表达基因,其表达量随着pH的升高而降低,pH为3.0时其表达量为对照条件下的10倍,pH为8.0时其表达量为对照条件下的0.36倍。柑橘果胶能够诱导PdPG2的表达,其表达量为对照的3.6倍。因此,在侵染过程中PdPG2表达的升高是由于发病部位酸化以及橘皮降解物诱导共同引起的。     

微重力对扩展青霉孢子萌发、菌丝生长和致病力的影响

 After being onto 22nd recoverable satellite of China for 18-day spaceflight under microgravity condition,me growth and pathogenicity of PeniciUium expansum were investigated.Spore germination rate of the spaceflight pathogen was insignificantly lower than that of the ground control.After germination,germ tube elongation of spaceflight pathogen was slower,as well as mycelia growth.However,there was no significant difference according to independent-samples T-test.The consistent results were obtained in vivo.The space. flight pathogen exhibited a little weaker pathogenicity in peach fruit.These findings suggested that the microgravity reduced the growth and pathogenicity of P.expansum.but the effect Was not marked.     

Characterizing the mechanism of biological control of postharvest diseases on fruits with a simple method to study competition for nutrients

ABSTRACT Biocontrol agents may compete with pathogens for nutrients and space to delay or prevent decay of fruits after harvest. These mechanisms of biological control have been difficult to study because no method has been available to determine the significance of each of the components of competition. We developed a nondestructive method using tissue culture plates with cylinder inserts containing defusing membrane at one end to study competition for nutrients without competition for space. Other biocontrol mechanisms in which direct contact between an antagonist and a pathogen is not required also can be studied. The method was used to determine the competition between the yeastlike biocontrol agent, Aureobasidium pullulans, and Penicillium expansum for limited nutrients in apple juice during 24 h incubation, simulating a fruit wound. The antagonist depleted amino acids and inhibited germination of P. expansum conidia. Exposing these conidia to fresh apple juice increased conidial germination to the level comparable to that exhibited by conidia which were not exposed to the antagonist. Because the culture plate method was nondestructive, follow-up experiments in an agar diffusion test were conducted. Juice in which the antagonist grew did not inhibit germination of P. expansum conidia that were seeded on the plates. This corroborates findings from the culture plate method that inhibition of the conidia germination resulted from competition for nutrients. The new method can be coupled with existing techniques to improve understanding of antagonist-pathogen interaction for biological control of postharvest diseases.     

Induction of Resistance to Penicillium digitatum in Grapefruit by the Yeast Biocontrol Agent Candida oleophila

ABSTRACT The yeast Candida oleophila, the base of the commercial product Aspire, is recommended for the control of postharvest decay in citrus and pome fruit. Its modes of action include nutrient competition, site exclusion, and direct mycoparasitism. In the present study, we showed that application of Candida oleophila to surface wounds or to intact 'Marsh Seedless' grapefruit elicited systemic resistance against Penicillium digitatum, the main postharvest pathogen of citrus fruit. The induction of pathogen resistance in fruit was already pronounced 24 h after elicitation; it was distance, concentration, and time dependent and restricted to the peel tissue closely surrounding the yeast application site. The induction of pathogen resistance required viable yeast cells at concentrations of 10(8) to 10(9) cells ml(-1). Nonviable autoclaved or boiled yeast cells or lower yeast concentrations were ineffective in enhancing fruit disease resistance. Application of Candida oleophila cell suspensions to grapefruit peel tissue increased ethylene biosynthesis, phenylalanine ammonia lyase activity, and phytoalexin accumulation, and increased chitinase and beta-1,3-endoglucanase protein levels, indicated by western immunoblotting analysis. Scanning electron microscope observations revealed that spore germination and germ tube growth of Penicillium digitatum were markedly inhibited in wounds made near the yeast-treated sites. Overall, this study provides evidence that induced resistance against postharvest decay of citrus fruit should be considered an important component of the multiple modes of action of the yeast Candida oleophila.     

Factors affecting UV-induced resistance in grapefruit against the green mould decay caused by Penicillium digitatum

Exposure of harvested grapefruit to ultraviolet (UV) light induced resistance against the green mould decay caused by Penicillium digitatum. Grapefruit picked at various times during the harvest season responded differently to UV treatments. The UV dose required for development of maximum resistance increased as the season progressed. The initial UV dose required in November-picked fruit for maximum response was 4·8 kJ/m². It declined to 1·6 and 3·2 kJ/m² in December- and January-picked fruit, respectively, and increased to 8 kJ/m² in February-picked fruit. Correspondingly, the minimum percentage infection developing after UV treatment increased throughout the season from 0 to 35%. Resistance in UV-treated fruit developed to its maximum extent at 24–48 h following exposure to UV light and then decreased. Development of induced resistance in grapefruit peel was affected by the temperature at which the fruit was stored 24 h after UV treatment and before infection with P. digitatum. In the UV-treated fruit, the fungus developed a sporadic mycelium with marked inhibition of sporulation. The activity of phenylalanine ammonia-lyase and peroxidase markedly increased in the peel following exposure of the fruit to UV light.     

Characterization of fludioxonil-resistant and pyrimethanil-resistant phenotypes of Penicillium expansum from apple

Penicillium expansum is the primary cause of blue mold, a major postharvest disease of apple. Fludioxonil and pyrimethanil are two newly registered postharvest fungicides for pome fruit in the United States. To evaluate the potential risk of resistance development in P. expansum to the new postharvest fungicides, one isolate of each of thiabendazole-resistant (TBZ-R) and -sensitive (TBZ-S) P. expansum was exposed to UV radiation to generate fungicide-resistant mutants. Four fludioxonil highly-resistant mutants (EC(50) > 1,000 microg/ml) and four pyrimethanil-resistant mutants (EC(50) > 10 microg/ml) were tested for sensitivities to thiabendazole, fludioxonil, and pyrimethanil, and fitness parameters including mycelial growth, sporulation on potato dextrose agar (PDA), sensitivity to osmotic stress, and pathogenicity and sporulation on apple fruit. The stability of resistance of the mutants was tested on PDA and apple fruit. Efficacy of the three fungicides to control blue mold incited by the mutants was evaluated on apple fruit. Six fungicide-resistant phenotypes were identified among the parental wild-type isolates and their mutants based upon their resistance levels. All four fludioxonil highly-resistant mutants were sensitive to pyrimethanil and retained the same phenotypes of resistance to TBZ as the parental isolates. All four pyrimethanil-resistant mutants had a low level of resistance to fludioxonil with a resistance factor >15. The two pyrimethanil-resistant mutants derived from a TBZ-S isolate became resistant to TBZ at 5 microg/ml. After 20 successive generations on PDA and four generations on apple fruit, the mutants retained the same phenotypes as the original generations. All mutants were pathogenic on apple fruit at both 0 and 20 degrees C, but fludioxonil highly-resistant mutants were less virulent and produced fewer conidia on apple fruit than pyrimethanil-resistant mutants and their parental wild-type isolates. Compared with the parental isolates, all four fludioxonil highly-resistant mutants had an increased sensitivity to osmotic stress on PDA amended with NaCl, while the pyrimethanil-resistant mutants did not. Pyrimethanil was effective against blue mold caused by fludioxonil-resistant mutants at both 0 and 20 degrees C. Pyrimethanil and fludioxonil reduced blue mold incited by pyrimethanil-resistant mutants during 12-week storage at 0 degrees C but were not effective at 20 degrees C. TBZ was not effective against pyrimethanil-resistant mutants derived from TBZ-S wild-type isolates at room temperature but provided some control at 0 degrees C. The results indicate that: (i) a fitness cost was associated with fludioxonil highly resistant mutants of P. expansum in both saprophytic and pathogenic phases of the pathogen but not pyrimethanil-resistant mutants; (ii) pyrimethanil possessed a higher risk than fludioxonil in the development of resistance in P. expansum; and (iii) triple resistance to the three apple-postharvest fungicides could emerge and become a practical problem if resistance to pyrimethanil develops in P. expansum populations.     

Influence of CaCl(2) on Penicillium digitatum, Grapefruit Peel Tissue, and Biocontrol Activity of Pichia guilliermondii

ABSTRACT Interactions between CaCl(2), grapefruit peel tissue, Penicillium digitatum, and the yeast antagonist Pichia guilliermondii strain US-7 were investigated. Application of 68 or 136 mM CaCl(2) to grapefruit surface wounds reduced the incidence of green mold caused by Penicillium digitatum by 43 to 52%. In laboratory tests, a cell suspension (10(7) cells/ml) of Pichia guilliermondii containing either 68 or 136 mM CaCl(2) reduced the incidence of green mold from 27 to 3%. In large scale tests, dip application of 136 mM CaCl(2) with US-7 (10(7) cells/ml) significantly decreased the number of wounds infected by Penicillium digitatum. CaCl(2), with or without yeast cells, stimulated ethylene production in grapefruit tissue. Increasing concentrations of CaCl(2) resulted in decreased spore germination and germ tube elongation of Penicillium digitatum. Pectinolytic activity of crude enzyme preparations of Penicillium digitatum was also inhibited by the presence of increasing concentrations of CaCl(2). US-7 exhibited a strong ability to maintain cytosolic Ca(2+) homeostasis at levels that did not exceed 1.4 muM when exposed to 150 mM CaCl(2). On the other hand, strain 114 of Debaryomyces hansenii, which failed to give any protection against infection by Penicillium digitatum, showed reduced capacity to maintain Ca(2+) homeostasis. The effect of calcium in reducing infection of grapefruit wounds by Penicillium digitatum could be due to direct effects on host tissue (making cell walls more resistant to enzymatic degradation) or the pathogen (interfering with spore germination, growth, and inhibition of fungal pectinolytic enzymes). Alternatively, the ability of US-7 to maintain calcium homeostasis may allow it to grow or assist in its competitive ability in a microenvironment that, because of high levels of calcium ions, is inhibitory to growth of the green mold pathogen.     

柑桔绿霉病拮抗细菌的筛选、鉴定及其抑制效果

从采集的柑桔园根际土壤中筛选得到1株对柑桔绿霉菌Penicillium digitatum具有较强拮抗活性的菌株HC-03.该菌株对柑桔绿霉菌、柑桔青霉菌Penicillium italicum、柑桔酸腐菌Geotrichum candidum等12种水果病原菌均有不同程度的拮抗作用,表现出广谱抗菌活性.菌落形态观察、生理生化特性分析及16S rDNA 序列测定和特异性PCR检测结果表明,该菌株为解淀粉芽孢杆菌Bacillus amyloliquefaciens.对沙糖桔的活体接种试验表明,菌株HC-03的发酵液、菌悬液及发酵滤液处理柑桔绿霉病的发生率均低于20%,病斑直径均在10mm以下.     

Evaluation of food additives and low-toxicity compounds as alternative chemicals for the control of Penicillium digitatum and Penicillium italicum on citrus fruit

The effectiveness of low-toxicity chemicals as possible alternatives to synthetic fungicides for the control of post-harvest green and blue moulds of citrus was evaluated. A preliminary selection of chemicals, mostly common food additives, was made through in vivo primary screenings with oranges artificially inoculated with Penicillium digitatum or P italicum. Selected compounds and mixtures were tested as heated solutions in small-scale trials. Immersion of artificially inoculated oranges or lemons for 120 s in solutions at 40.6 degrees C and natural pH of potassium sorbate (0.2 M), sodium benzoate (0.2 M) or mixtures (0.1 + 0.1 M) of potassium sorbate with sodium benzoate, sodium propionate or sodium acetate were the most effective organic acid salts tested and reduced green mould by 70-80% after 7 days of storage at 20 degrees C. The mixtures did not significantly enhance the effectiveness of potassium sorbate or sodium benzoate alone. These solutions were as effective as sodium carbonate or calcium polysulphide treatments and, in general, they were more effective on lemons than on oranges. Satisfactory control of green and blue moulds was obtained by dipping oranges for 150 s in solutions of sodium molybdate (24.2 mM) or ammonium molybdate (1.0 mM) at 48 or 53 degrees C, but not at 20 degrees C. At 53 degrees C, however, the effectiveness of hot water was not enhanced by either molybdate. Molybdenum salts at higher concentrations were phytotoxic and stained the fruit. At non-phytotoxic concentrations, the effectiveness of these solutions was more influenced by temperature than by concentration. In general, the inhibitory effects of all compounds tested were not fungicidal but fungistatic and not very persistent. In conclusion, potassium sorbate, sodium benzoate and ammonium molybdate, among the wide range of chemicals tested, were superior for the control of post-harvest Penicillium decay of citrus fruit.     

Cell wall-degrading enzymes of Didymella bryoniae in relation to fungal growth and virulence in cantaloupe fruit

Didymella bryoniae is an important pathogen of cucurbits worldwide. Virulence factors of D. bryoniae were investigated in regard to fungal growth and the production of the cell wall-degrading enzymes, polygalacturonase (PG), pectate lyase (PL), pectin lyase (PNL), β-galactosidase (β-Gal) and cellulase (Cx). Virulence levels of five D. bryoniae isolates were determined by the severity of inoculated cantaloupe fruit decay. The highly virulent isolates had more mycelial growth than the moderately virulent isolates in different media. PG activities produced by the highly virulent isolates in shake cultures and in decayed fruit were greater than those of the moderately virulent isolates. PNL, but not PL, in decayed fruit was higher with the highly virulent isolates compared to the moderately virulent ones. The highly virulent isolates showed higher Cx activity than the moderately virulent ones in decayed fruit and in fruit tissue shake culture. β-Gal activities of the highly virulent isolates in pectin shake culture and in decayed fruit were greater than those of the two moderately virulent isolates although fruit also produced β-Gal. Protein analysis showed two fungal β-Gal isozymes in decayed fruit compared to those of healthy fruit. Correlation analysis indicated that the activities of PG, PNL, β-Gal and Cx in cultures and in decayed fruit positively correlated with fungal growth and fruit decay severity. The results of this study suggest that PG, PNL, β-Gal, and Cx appear to be virulence factors of D. bryoniae in cantaloupe decay with PG and β-Gal as the most predominant fruit decay enzymes.     

Sensitivity of Penicillium digitatum and P. italicum to Postharvest Citrus Fungicides in California

ABSTRACT Penicillium digitatum isolates (326), collected in California citrus groves and packinghouses, were assayed qualitatively for their sensitivity to imazalil, thiabendazole, and o-phenylphenol. Eighteen typical triple-resistant isolates, acquired in each of 3 years (1988, 1990, and 1994), were assayed quantitatively for their sensitivity to each of the three fungicides. No significant differences were found in the mean sensitivity of the isolates collected in different years. However, the proportion of isolates that were resistant to all three fungicides increased from 43% in 1988 to 77% in 1990 and 74% in 1994. Imazalil-resistant biotypes of P. digitatum were isolated frequently in California packinghouses, while resistant P. italicum was rare. No fungicide-resistant biotypes of either species were collected from citrus groves. Wild-type P. italicum was slightly less sensitive than wild-type P. digitatum to all three fungicides. The concentration of imazalil producing 50% growth inhibition (EC(50)) was three times greater when the age of the P. digitatum assay inoculum was increased from 12 to 24 h. Activity of imazalil increased with pH of the assay medium in the range pH 5.1 to 5.9, reflecting the greater concentration of dissociated imazalil at the higher pH value.     

Control of postharvest pathogens and colonization of the apple surface by antagonistic microorganisms in the field

ABSTRACT Selected isolates of Aureobasidium pullulans, Rhodotorula glutinis, and Bacillus subtilis reduced the size and number of lesions on wounded apples caused by the postharvest pathogens Penicillium expansum, Botrytis cinerea, and Pezicula malicorticis. Combinations of the antagonistic microorganisms were applied to apple trees in the field late in the growing season of two consecutive years. The population dynamics of the introduced microorganisms and the incidence of fruit decay were determined. Population sizes of introduced antagonists on apple surfaces increased in the field following application of treatments until harvest. After transfer of the fruit from the field into cold storage, the populations of the introduced antagonists remained higher than in the control treatments. Identification of the applied isolates of A. pullulans and R. glutinis during the experiments was achieved by isolate-specific DNA probes generated from random amplified polymorphic DNA. A combination of two strains of A. pullulans and one strain of R. glutinis suppressed rotting of apple to the same extent as the commonly used fungicide Euparen. Our data demonstrate that the application of antagonistic microorganisms in the field represents a promising alternative to fungicide treatments to control post-harvest diseases of apple.     

Fusarium solani endo-polygalacturonase from decayed muskmelon fruit: purification and characterization

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. TheK_m and V_maxof PG1 using polygalacturonic acid as the substrate were 1.34 mg ml^-1and 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 .     

Induction of Chitinase and β-1,3-Endoglucanase Proteins by UV Irradiation and Wounding in Grapefruit Peel Tissue

UV irradiation enhanced the resistance of grapefruit against the development of green mold décay caused byPenicillium digitatum, the main postharvest pathogen of citrus fruit, and significantly inhibited the fungus’ growth at the fruit wound sites. Immunoblotting analysis using specific citrus chitinase and β-1,3-endoglucanase antibodies, showed that UV irradiation, wounding of the fruit, or a combination of these two treatments, induced the accumulation of a 25 kD chitinase protein in the fruit’s peel tissue. On the other hand, UV irradiation or wounding of the fruit alone was unable to induce the accumulation of 39 and 43 kD β-1,3-endoglucanase proteins, but the combination of the two treatments increased these protein levels. It is suggested that both chitinase and β-1,3-endoglucanase may play a role in the UV-induced resistance of grapefruit againstP. digitatum. Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 403/99. http://www.phytoparasitica.org posting June 3, 1999.