Effect of octanal on the mycelial growth of Penicillium italicum and P. digitatum

The present study investigated the antifungal activity of octanal against Penicillium italicum and P. digitatum. Results showed that octanal exhibited strong antifungal activity against the test pathogens in a dose-dependent manner. Scanning electron microscopy observation revealed that octanal obviously altered the morphology of P. italicum and P. digitatum hyphae by causing the loss of cytoplasm and distortion of mycelia. A rapid increase in the membrane permeability of P. italicum and P. digitatum was observed after treated with octanal at minimum inhibitory concentration or minimum fungicidal concentration, evidenced by the release of cell constituents, the extracellular conductivity and the extracellular potential of hydrogen. In addition, octanal apparently induced a decrease in total lipid contents of P. italicum and P. digitatum cells. These results suggested that the antifungal activity of octanal against P. italicum and P. digitatum can be attributed to the disruption of the cell membrane integrity and the leakage of cell components.     

Sulla penetrazione di Penicillium digitatum Sacc. e Penicillium italicum Wehmer nei frutti degli agrumi

Summary

The A., with a series of controls and investigations, accomplished on different groups of Oranges and following various forms of artificial infection with Penicillium digitatum and Penicillium italicum, renders evident the different reactions that these moulds have with regard to the resistance of the cuticle and the epidermic and under-epidermic stratums of the peel of the controlled fruits. Moreover the A. confirms that both the P. digitatum and the P. italicum can act as «parasite of injury» and as «parasite of contact».     

Potential for Development of Tolerance by Penicillium digitatum and Penicillium italicum after Repeated Exposure to Potassium Sorbate

Two strains of Penicillium digitatum and one strain of Penicillium italicum were exposed to various levels of sorbic acid and potassium sorbate, and the MICs were determined. Selected strains of the molds were then repeatedly exposed to subinhibitory levels of the compounds to determine whether increased tolerance might develop. The MIC of sorbic acid (pH 4.75) to P. digitatum was between 0.02 and 0.025%. The MIC of sorbate (pH 5.5) to two strains of P. digitatum and P. italicum was found to be between 0.06 and 0.08%. Increasing levels of sorbate resulted in increasing growth suppression of the molds. Populations of P. digitatum were tested for increased tolerance to sorbic acid, and none was found. Individual molds that started from the same parent colony were examined for increased tolerance to potassium sorbate. Two P. digitatum strains developed no observable increased tolerance, but P. italicum developed a slight increase in tolerance to sorbate. When spores of P. italicum and P. digitatum were exposed to higher levels of sorbate for prolonged times, the fungicidal or fungistatic activity of the inhibitor was dependent upon pH, length of exposure time, level of sorbate, and the mold strain.     

Effect of water activity and temperature on germination and growth of Penicillium digitatum,P. italicum and Geotrichum candidum

AIMS: This study compares the effect of temperature (4-37 degrees C) and water activity (aw: 0.99-0.87) and their interactions on the germination rates, lag times prior to germination and mycelial growth 'in vitro' of Penicillium digitatum, P. italicum and Geotrichum candidum, the main postharvest pathogens affecting citrus fruits. METHODS AND RESULTS: Germination and growth were markedly influenced by temperature and aw. Generally, lag times were longer and germination and growth rates were slower when conditions of temperature and aw were far from optimum. All the studied species were able to germinate over a range of 4-30 degrees C at 0.995 aw, although in non-optimal conditions P. digitatum only reached 40-60% of germinated conidia. At low temperatures, P. italicum germinated and grew faster than P. digitatum and G. candidum, particularly at 0.95 aw. Penicillium italicum was also able to germinate and grow in the driest studied conditions (0.87 aw), while G. candidum did not germinate under 0.95 aw. CONCLUSIONS: Knowledge of the ecological requirements of these fungi is important in order to understand their behaviour in natural situations and to predict fungal spoilage on citrus fruits.     

In vitro studies on the effect of some chemicals on the growth and sporification of Penicillium digitatum and P. italicum

The behaviour of Penicillium digitatum and P. italicum was investigated when subjected to different concentrations of methanol (MeOH) and dimethyl sulfoxide (DMSO). The experiments were performed in 9 cm Petri dishes containing PDA amended with 0, 5, 10, 20, 30, 40 or 50 microL/mL of each of the single or combined chemicals. Daily, the formed colonies (cfu), the colony diameter and the degree of sporification were monitored during incubation at 20 degrees C for 5 day. Additionally, the pathogen development and its performance were studied by scanning electron microscopy (SEM). According to the chemical, the mycelium growth rate was affected differently and, compared to the control, only MeOH inhibited the expansion of the colony diameter. This effect was more pronounced for P. italicum. A nearly linear drop of cfu was observed as the concentration of the two chemicals was raised, and a complete inhibition of the two pathogens was attained with 50 microL/mL MeOH. With respect to the sporification degree the two pathogens were influenced similarly, but the tested compounds had opposite effects. Indeed, with MeOH, sporification took place earlier (24-36 h postinoculation) compared to the control (60 h), while during the whole experiment, DMSO at concentrations higher than 0.5 microL/mL, drastically inhibited the sporification. SEM observations of P. digitatum growth on DMSO amended media evidenced a marked increase of mycelium branching and alterations to the conidiophore, while MeOH reduced the mycelium length and fastened the conidiophore formation. The combination of the two compounds produced a synergistic interaction reducing by 40% the concentration required to inhibit completely the germination and growth of P. digitatum.     

寡雄腐霉发酵液的动物毒性及其对柑橘果实贮藏期青、绿霉病的防治效果

摘要:【目的】研制安全、无毒、高效的生物保鲜剂,降低意大利青霉(Penicillium italicum,青霉)和指状青霉(Penicillium digitatum,绿霉)引起的柑橘烂果。【方法】试验利用自主选育的寡雄腐霉优良菌株(Pythium oligandrum CQ2010),制备发酵液,测试了对小鼠的急性毒性,并设置对照(液体培养基,CK)、寡雄腐霉发酵液(P． oligandrum Broth,POB)、咪鲜胺(Prochloraz,PC)、咪鲜胺+ POB(PC+POB)等4种处理,研究了它们对青、绿霉菌的抑制作用及其对柑橘防腐保鲜的作用。【结果】用大剂量的POB灌胃给药对小鼠体重增长无显著影响,供试动物的外观和行为均无异常,心、肝、肾、肺、肠等组织器官也未见病理改变。POB显著抑制青、绿霉菌丝生长和孢子萌发,抑制率分别为70.24%－93.74%(菌丝生长)和44.91%－87.82%(24 h孢子萌发)。柑橘果实接种青霉后,烂果率CK＞POB、PC＞PC+POB,防治效果PC+POB＞POB、PC。在模拟柑橘商品化贮藏保鲜试验中,青、绿霉发病率占总发病率的50%以上,CK、POB、PC和PC + POB的烂果率依次为26.40%、15.03%、16.61%和4.21%。此外,POB对果实品质无显著影响,但显著提高果皮中的超氧化物歧化酶、过氧化物酶和过氧化氢酶活性,有益于提高柑橘果实的抗病性和贮藏性。【结论】在柑橘贮藏过程中,POB对果实青、绿霉病有显著的防治作用,并与咪鲜胺的防病效果有叠加作用。     

In vitro effects of water activity, temperature and solutes on the growth rate of P. italicum Wehmer and P. digitatum Sacc

AIMS: To evaluate the effect of water activity (a(w) 0.98-0.89, adjusted with glycerol, sorbitol, glucose, or NaCl) and temperature (5-25 degrees C) on the lag phase and radial growth rate (mm day(-1)) of the important citrus spoilage fungi, such as Penicillium italicum and Penicillium digitatum grown in potato dextrose agar (PDA) medium. To select, among models based on the use of different solutes, a model fitting accurately the growth of these species in relation to a(w) and temperature. METHODS AND RESULTS: Extensive data analyses showed for both Penicillium species a highly significant effect of a(w), temperature, solutes and their interactions on radial growth rate (P < 0.0001). Radial growth rate was inhibited and the lag phase (i.e. the time required for growth) lengthened as the a(w) of the medium decreased. NaCl appeared to causes the greatest stress on growth when compared with other nonionic solutes. Penicillium italicum stopped growing at 0.96 a(w) and P. digitatum at 0.93 a(w). Under the dry conditions where growth was observed, P. italicum grew faster than P. digitatum at low temperature and P. digitatum remained more active at ambient temperature. Multiple regression analysis applied to the square roots of the growth rates observed in the presence of each solute showed that both the 'glycerol model' and the 'sorbitol model' yielded a good prediction of P. italicum growth and the 'sorbitol model' gave an accurate fit for P. digitatum growth, offering high-quality prediction within the experimental limits described. CONCLUSIONS: Mathematical models describing and predicting, as a function of a(w) and temperature, the square root of the radial growth rate of the agents responsible for blue and green decays are important tools for understanding the behaviour of these fungi under natural conditions and for predicting citrus fruit spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of these results should contribute towards a more rational control strategy against citrus spoilage fungi.     

Production and catabolite repression of Penicillium italicum beta-glucanases.

The filamentous fungus Penicillium italicum, grown in a defined liquid medium, produced beta-1,3-glucanase, which remained essentially bound to the cells, and beta-1,6-glucanase, an essentially extracellular enzyme. When glucose was depleted from the medium, when a limited concentration of glucose (0.2%) was maintained, or when the carbon source was galactose (3%) or lactose (3%), a significant increase in the specific activity of beta-1,3-glucanase, in cell extracts, took place. This was paralleled by a very slow rate of growth, and under glucose limitation, the appearance of beta-1,3-glucanase in the medium was also observed. On the other hand, when an excess of glucose, fructose, or sucrose was present, the specific activity remained constant and active growth was promoted. Laminarin, cellobiose, gentiobiose, and isolated Penicillium italicum walls were not capable of significantly inducing beta-1,3-glucanase synthesis to a level beyond that attained by glucose limitation. A similar behavior was observed for beta-1,6-glucanase. beta-1,3-Glucanase and beta-1,6-glucanase are therefore constitutive enzymes subjected to catabolite repression. The results are discussed in the context of the possible functions that have been suggested for glucanases and related enzymes.     

Geraniol biotransformation-pathway in spores of Penicillium digitatum

Spores of Penicillium digitatum ATCC 201167 transform geraniol, nerol, citral, and geranic acid into methylheptenone. Spore extracts of P. digitatum convert geraniol and nerol NAD+-dependently into citral. Spore extract also converts citral NAD+-dependently into geranic acid. Furthermore, a novel enzymatic activity, citral lyase, which cofactor-independently converts citral into methylheptenone and acetaldehyde, was detected. These result show that spores of P. digitatum convert geraniol via a novel biotransformation pathway. This is the first time a biotransformation pathway in fungal spores has been substantiated by biochemical studies. Geraniol and nerol are converted into citral by citrol dehydrogenase activity. The citral formed is subsequently deacetylated by citral lyase activity, forming methylheptenone. Moreover, citral is converted reversibly into geranic acid by citral dehydrogenase activity.     

Inhibition of Ethylene Production in Penicillium digitatum

Production of ethylene by static cultures of Penicillium digitatum, which utilize glutamate and α-ketoglutarate as ethylene precursors, was inhibited by methionine, methionine sulfoxide, methionine sulfone, and methionine sulfoximine. Rhizobitoxine did not affect ethylene production but its ethoxy and methoxy analogues were effective inhibitors of ethylene production; its saturated methoxy analogue and kainic acid stimulated ethylene production. Tracer studies showed that the inhibitors blocked the conversion of [³H]glutamate into [³H]ethylene.

In shake cultures of this fungus, which utilize methionine as the ethylene precursor, rhizobitoxine and its unsaturated analogues all inhibited, while the saturated methoxy analogue stimulated ethylene production. In both types of cultures inhibition was irreversible and was diminished by increasing concentrations of the ethylene precursor. The inhibitory activity or lack of it by rhizobitoxine and its analogues appears to be a function of their structural resemblance to glutamate and methionine as well as of their size and stereoconfiguration. These data suggest similarities between the ethylene-forming system in the fungus and in higher plants despite differences in precursors under some cultural conditions.

     

The biogenesis of ethylene in Penicillium digitatum

The origin of ethylene in Penicillium digitatum has been shown to be intimately associated with the Krebs cycle. 2-Ketoglutaric acid and glutamic acid are the most efficient precursors of ethylene, which is derived from carbons 3 and 4 of these substrates as a unit. However, which of these is the immediate precursor has not been established. Since 2-ketoglutaric acid is a very efficient precursor and succinic acid is an inefficient one, 2-ketoglutaric acid must be the branching point at which the pathway of ethylene biosynthesis leads away from the Krebs cycle. This conclusion is in full agreement with the following observations: Three of the four hydrogen atoms of the ethylene molecule were derived from protons of the medium; C-2 but not C-1 of acetate was incorporated into ethylene; and [2,3-¹⁴C]succinic acid but not [2,3-³H]succinic acid was incorporated.     

Regulation of beta-1,3-glucanase synthesis in Penicillium italicum.

The filamentous fungus Penicillium italicum produced a certain level of beta-1,3-glucanase during active growth in a glucose-supplemented medium; however, at a low glucose concentration (2 to 10 mM), derepression took place and the specific activity of the enzyme increased significantly. Derepressed cells (incubated in a glucose-limited medium) accumulated a capacity for the synthesis of beta-1,3-glucanase, which led to a subsequent increase in the specific activity even when the cells were transferred to a medium with an excess of glucose (180 mM). Two protein synthesis inhibitors, cycloheximide and trichodermin, immediately stopped the increase in specific activity when added to derepressed cells. On the other hand, 8-hydroxyquinoline, an RNA a synthesis inhibitor, acted differently, since it permitted the specific activity to increase for some time after being added to depressed cells. Moreover, the concentration of glucose did not affect the 8-hydroxyquinoline-insensitive synthesis of beta-1,3-glucanase. It is concluded that the glucose repression effect on beta-1,3-glucanase production must be exerted at a pretranslational level that could be either mRNA synthesis or some stage of the process involved in its maturation or stabilization.     

Toxicity of terpenes to spores and mycelium of Penicillium digitatum

Spores, although often considered metabolically inert, catalyze a variety of reactions. The use of spores instead of mycelium for bioconversions has several advantages. In this paper, we describe the difference in susceptibility of mycelium and spores against toxic substrates and products. A higher resistance of spores toward the toxic effects of bioconversion substrates and products is an advantage that has not been studied in detail until now. This paper shows that spores of Penicillium digitatum ATCC 201167 are on average over 2.5 times more resistant than mycelium toward the toxicity of substrates, intermediates, and products of the geraniol bioconversion pathway. Furthermore, the higher resistance of spores to citral was shown as an advantage in its biotransformation by P. digitatum. Using three different approaches the toxicity of the compounds were tested. The order of toxicity toward P. digitatum was, starting with the most toxic, citral > nerol/geraniol > geranic acid > methylheptenone > acetaldehyde.     

On the Roles of Calcineurin in Fungal Growth and Pathogenesis

Calcineurin is a calcium-activated phosphatase that controls morphogenesis and stress responses in eukaryotes. Fungal pathogens have adopted the calcineurin pathway to survive and effectively propagate within the host. The difficulty in treating fungal infections stems from similarities between pathogen and host eukaryotic cells. Using calcineurin inhibitors such as cyclosporin A or tacrolimus (FK506) in combination with antifungal drugs, including azoles or echinocandins, renders these drugs fungicidal, even towards drug-resistant species or strains, making calcineurin a promising drug target. This article summarizes the current understanding of the calcineurin pathway and its roles in governing the growth and virulence of pathogenic fungi, and compares and contrasts the roles of calcineurin in fungal pathogens that infect humans (Candida albicans and Cryptococcus neoformans) or plants (Magnaporthe oryzae and Ustilago maydis). Further investigation of calcineurin biology will advance opportunities to develop novel antifungal therapeutic approaches and provide insight into the evolution of virulence.     

Regulation of ethylene production by phosphate in Penicillium digitatum

Inorganic phosphate regulated ethylene production in shake culturesof Penicillium digitatum. Decreasing the phosphate level ofthe medium from 100 to 0.01 mM markedly increased, about 100-fold,the rate of ethylene production, in 96 hr, which was confinedentirely to the fungal mycelium. Exogenous addition of between0.01 to 100 mM phosphate, to high ethylene producing, low-phosphatecultures strongly inhibited their ethylene production and increasedthe ATP content of the mycelium. Phosphate also inhibited ethyleneproduction in apple slices. Addition of calcium ions to theincubation medium stimulated the production of ethylene in appleslices, subhook epicotyl segments of pea and shake culturesof P. digitatum. We suggest that this stimulatory effect wascaused by the reduction of inhibitory levels of phosphate, whichcomplexed with calcium. Thus, phosphate in conjunction withcalcium may play an important role in regulating ethylene productionnot only in P. digitatum but also in higher plants. ¹ On leave from the Agricultural Research Organization, TheVolcani Center, Israel. ² On leave from the M.S. University of Baroda, India. (Received September 7, 1977; )