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.     

赣南脐橙绿霉病菌对常用杀菌剂抗性监测

 本文研究了来自赣南7个县的柑橘绿霉病菌(Penicillium digitatum)种群对该地区常用杀菌剂抑霉唑、咪鲜胺、甲基硫菌灵和百可得的抗性频率、抗性水平和对抑霉唑的抗性分子机制。结果表明:病菌对抑霉唑和咪鲜胺存在基本一致的抗性;2011和2012年病菌种群对抑霉唑和咪鲜胺的抗性频率分别为82%和90%,平均抗性倍数为51.5倍,抗性分子机制均属于IMZ-R3,即CYP51B基因启动子区发生199 bp插入的突变;病菌种群对甲基硫菌灵的抗性频率分别为82%和91%;病菌种群对百可得均表现敏感。本研究为采后柑橘病害防治药剂选择提供了科学的依据。     

Liquid Formulation of the Postharvest Biocontrol Agent Candida sake CPA-1 in Isotonic Solutions

ABSTRACT Viability of the postharvest biocontrol agent Candida sake CPA-1 stored as liquid formulation was evaluated by studying the effect of growth, preservation medium, and temperature. C. sake was grown in molasses medium with unmodified water activity (a(w)) and in the same with a(w) modified to 0.98 with the addition of several solutes. Cells were preserved with isotonic solutions of different substances. Efficacy of liquid formulations stored for different periods was tested against infection by Penicillium expansum on apples. The best growth media were the (unmodified one and those modified to 0.98 a(w) with the addition of glycerol or sorbitol. For all growth media, the best preservation medium was the isotonic solution prepared with trehalose. When the effect of trehalose concentration in the preservation medium was studied, generally, at trehalose concentrations below the isotonic one, C. sake viabilities increased with increased trehalose. However, the best results were obtained when cells were preserved with the trehalose solution which was isotonic with cells. After 7 months of storage at 4 degrees C, cells that were grown in the sorbitol-modified medium and preserved with the isotonic solution of trehalose (0.96 M) maintained their viability and efficacy against P. expansum infection of apples.     

The ultrastructure of germinating conidia of Penicillium digitatum inhibited by sec-butylamine

Electron micrographs of germinating conidia of P. digitatum showed osmiophilic inclusions scattered in the ground plasm or located within membrane-bound vacuoles where they were associated with various membranes and “myelin-like” structures. Cytochemical studies suggested that the osmiophilic inclusions within the vacuoles contained phospholipids and proteins. These phospholipoprotein inclusions may provide precursors needed for the assembly of membrane-bound organelles formed during germination. In conidia germinated in the presence of fungistatic concentrations of sec-butylamine (SBA) the vacuoles enlarged and the phospholipoprotein inclusions decreased in size and appeared to disintegrate and give rise to membrane-bound vesicles. SBA may deprive the conidium of materials normally utilized for membrane formation by the developing organelles.     

柑橘绿霉病菌木聚糖酶基因(PdXY2)功能初步研究

柑橘绿霉病菌(Penicillium digitatum)是引起柑橘储藏期腐烂最主要的病原之一,严重影响了柑橘产业发展。本试验克隆了柑橘绿霉病菌的木聚糖酶基因(PdXY2),对其表达进行研究,在柑橘发病过程中,PdXY2的表达显著升高,至48h达到最高,其表达量为对照的4倍。为进一步明确PdXY2基因功能,构建了PdXY2基因缺失突变株(ΔPdXY2),ΔPdXY2突变株的致病性与野生型相比没有明显差异。由此我们推断,在柑橘绿霉病菌侵染柑橘的过程中PdXY2有一定的作用,但是单一缺失木聚糖酶PdXY2基因不能影响其致病性。     

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.     

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.     

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。     

Evidence for Antibiosis and Induced Host Defense Reactions in the Interaction Between Verticillium lecanii and Penicillium digitatum, the Causal Agent of Green Mold

ABSTRACT Chronological events of the intercellular interaction between Verticillium lecanii and the postharvest pathogen Penicillium digitatum were investigated by transmission electron microscopy and gold cytochemistry. Growth inhibition of P. oligandrum as a response to V. lecanii attack correlated with striking host changes including retraction of the plasma membrane and cytoplasm disorganization. Such changes were associated with the deposition on the inner host cell surface of a chitin- and cellulose-enriched material which appeared to be laid down as a structural defense reaction. The accumulation of chitin in the newly formed material correlated with a decrease in the amount of wallbound chitin. However, the deposition of cellulose appeared to correspond to a de novo synthesis, as evidenced by the occurrence of cellulose-containing vesicles which released their content in the space between the invaginated plasma membrane and the host cell wall. Results of the present study provide the first ultrastructural and cytochemical evidence that antagonism, triggered by V. lecanii, is a multifaceted process in which antibiosis, with alteration of the host hyphae prior to contact with the antagonist, appears to be the key process in the antagonism against P. digitatum.     

Nutritional Enhancement of Biocontrol Activity of Candida sake (CPA-1) against Penicillium expansum on Apples and Pears

Pome fruits are poor in nitrogenous compounds and the addition of nitrogen can improve colonisation of the fruits by antagonists. Twenty-two nitrogenous compounds were evaluated for their effect on Candida sake (CPA-1) growth in vitro. Ten compounds that induced greater growth were applied with the antagonist to wounded fruits to evaluate their effect on enhancing control of Penicillium expansum. Calcium chloride and 2-deoxy-D-glucose were also tested. L-serine and L-aspartic acid enhanced biocontrol by C. sake against P. expansum on apples. On apples and pears, ammonium molybdate, calcium chloride and 2-deoxy-D-glucose improved the capacity of the antagonist to control P. expansum. The addition of ammonium molybdate at 1 mM allowed C. sake to be used on apples and pears at a lower concentration without diminishing control. Similar results were observed with the addition of calcium chloride to the antagonist. 2-deoxy-D-glucose at 6 and 18 mM enhanced biocontrol on pears by over 81%, but on apples the improvement of biocontrol was observed only at 6 mM. In cold storage, the combination of ammonium molybdate and C. sake completely eliminated the incidence of blue mould on pears, and reduced its severity and incidence by more than 80% on apples.     

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.     

Production, Survival, and Evaluation of Solid-Substrate Inocula of Penicillium oxalicum, a Biocontrol Agent Against Fusarium Wilt of Tomato

ABSTRACT Production of conidia of Penicillium oxalicum (ATCC number pending), a biocontrol agent of Fusarium oxysporum f. sp. lycopersici, was tested in liquid and solid fermentation. P. oxalicum produced 250-fold more conidia in solid than in liquid fermentation at 30 days after inoculation of substrate. Solid fermentation was carried out in plastic bags (600 cm(3)) especially designed for solid fermentation (VALMIC) containing 50 g of peat/vermiculite (PV) (1:1, wt/wt) with 40% moisture, sealed, sterilized, and then inoculated with 1 ml of a conidial suspension of P. oxalicum (10(5) conidia g(-1) dry substrate), sealed again, and incubated in darkness at 20 to 25 degrees C for 30 days. Addition of amendments to PV in a proportion of 0.5 (wt/wt) significantly increased conidial production of P. oxalicum. The best production was obtained on PV plus meal of cereal grains (barley) or leguminous seeds (lentil) (100-fold higher). Conidial production obtained after 5 days of inoculation was similar to that obtained at 30 days. However, viability of conidia produced in PV plus lentil meal was 35% higher than that of conidia produced in PV plus barley meal. Changes in proportions (1:1:0.5, wt/wt/wt; 1:1:1, wt/wt/wt; 1:0.5:0.5, wt/wt/wt; 1:1:0.5, vol/vol/vol) of components of the substrate (peat/vermiculite/lentil meal) did not enhance production or viability of conidia. Optimal initial moisture in the substrate was 30 to 40%. At lower moistures, significant reductions of production of conidia were observed, particularly at 10%. There was a general decline in the number of conidia in bags with time of storage at -80, -20, 4, and 25 degrees C, or at room temperature (range from 30 to 15 degrees C), with the highest decline occurring from 60 to 180 days. Conidial viability also was reduced with time, except for conidia stored at -20 degrees C. Fresh conidia produced in solid fermentation system or those conidia stored at -20 degrees C for 180 days reduced Fusarium wilt of tomato by 49 and 61%, respectively.     

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.     

寡糖类物质诱导黄瓜对霜霉病的抗性

霜霉病是黄瓜栽培中发生最为普遍的病害之一，目前生产上主要利用化学药剂来防治，存在着农药残留的现象，从而影响了无公害绿色食品的生产。寡糖是新一类生物诱抗剂，具有可自然降解，不污染环境，无残留等优点。本文以酶解方法从海带中获得寡糖类物质，利用其防治黄瓜霜霉病[Pseudoperonospora cubensis(Berk．et Curt．)Rostov．]取得了一定的效果。现将研究结果报道如下。     

Systemic Resistance Induced by Trichoderma spp.: Interactions Between the Host, the Pathogen, the Biocontrol Agent, and Soil Organic Matter Quality

ABSTRACT Several factors affect the ability of Trichoderma spp. to provide systemic disease control. This paper focuses on the role of the substrate in which plants are grown, resistance of the host to disease, and the ability of introduced Trichoderma inoculum to spread under commercial conditions. Several reports reveal that foliar disease control provided by Trichoderma spp. is more effective on plants grown in compost-amended media compared with in lower-in-microbial-carrying-capacity sphagnum peat media. In Rhododendron spp., host resistance affects control of Phytophthora dieback provided by Trichoderma spp. For example, T. hamatum 382 (T382) significantly (P = 0.05) suppressed the disease on susceptible cv. Roseum Elegans while plant vigor was increased. The disease was not suppressed, however, on highly susceptible cvs. Aglo and PJM Elite even though the vigor of these plants was increased. Using a strain-specific polymerase chain reaction assay under commercial conditions, it was demonstrated that introduced inoculum of T382 did not spread frequently from inoculated to control compost-amended media. Other Trichoderma isolates typically are abundant in control media within days after potting unless inoculated with a specific Trichoderma isolate. Thus, the low population of isolates that can induce systemic resistance in composting and potting mix environments may explain why most compost-amended substrates do not naturally suppress foliar diseases.     

UV irradiation Alters the Levels of Flavonoids Involved in the Defence Mechanism of Citrus aurantium Fruits against Penicillium digitatum

The effect of UV irradiation on the levels of the flavanone, naringin, and the polymethoxyflavone, tangeretin, in the peel of Citrus aurantium fruits is described, as changes in the synthesis and/or accumulation of these compounds after infection with Penicillium digitatum. The growth of P. digitatum on previously irradiated fruit was reduced by up to 45%. Changes in flavonoid levels were detected, associated with inhibition of fungus growth, the naringin content falling by 69% and tangeretin levels increasing by 70%. The possible participation of naringin and tangeretin in the defence mechanism of this Citrus species is discussed.