Effects of the origins of Botrytis cinerea on earthy aromas from grape broth media further inoculated with Penicillium expansum

Earthy “off” aromas from wine and grape juice are highly detrimental to the production of quality grape products. These volatile compounds are produced on grapes by Botrytis cinerea, Penicillium expansum and/or a combination of P. expansum and B. cinerea strains. B. cinerea strains were isolated from different (a) vineyards in Spain and Portugal, (b) grape varieties (c) bunches (i.e., sound and botrytized) and (d) positions in the botrytized bunch (i.e., interior or exterior). A novel Headspace-Phase Microextraction (SPME) followed by Gas Chromatrography/Mass Spectrometry (GC-MS) dedicated to analyze geosmin, methylisoborneol (MIB), 1-octen-3-ol, fenchone and fenchol in grape broth medium was used. Approximately 50% of the B. cinerea strains induced detectable geosmin. One strain accumulated significant amounts of anisoles, demonstrating that this contamination might already occur in the vineyard. Strains from the interior of Cainho grape bunches induced more geosmin and hence it may be possible to reduce this volatile in wine by avoiding using these grapes in case of B. cinerea attack.     

Yeast biocontrol of fungal spoilage of pears stored at low temperature

To reduce the use of fungicides, biological control with yeasts has been proposed in postharvest pears. Most studies of antagonists selection have been carried out at room temperature. However, in regions like North Patagonia where fruits are stored at − 1/0 °C during 5-7 months the selection of potential antagonist agents must be carried out at low temperature. In this study, 75 yeast cultures were isolated from healthy pears from two Patagonian cold-storage packinghouses. Aureobasidium pullulans, Cryptococcus albidus, Cryptococcus difluens, Pichia membranifaciens, Pichia philogaea, Rhodotorula mucilaginosa and Saccharomyces cerevisiae yeast species were identified. Additionally, 13 indigenous isolates of Penicillium expansum and 10 isolates of Botrytis cinerea were obtained from diseased pears, characterized by aggressiveness and tested for sensitivity to postharvest fungicides. The yeasts were pre-selected for their ability to grow at low temperature. In a first biocontrol assay using the most aggressive and the most sensitive isolate of each pathogen, two epiphytic isolates of A. pullulans and R. mucilaginosa were the most promising isolates to be used as biocontrol agents. They reduced the decay incidence by P. expansum to 33% and the lesion diameter in 88% after 60 days of incubation in cold. Foreign commercial yeast used as a reference in assays, only reduced 30% of lesion diameter in the same conditions. Yeasts were not able to reduce the incidence of B. cinerea decay. The control activity of the best two yeasts was compared with the control caused by the fungicides in a second bioassay, obtaining higher levels of protection against P. expansum by the yeasts. These two regional yeasts isolates could be promising tools for the future development of commercial products for biological control.     

Effect of chitin on the antagonistic activity of Rhodotorula glutinis against Botrytis cinerea in strawberries and the possible mechanisms involved

The influence of chitin on the efficacy of Rhodotorula glutinis in controlling postharvest grey mould of strawberries and the possible mechanisms involved were investigated. The results showed that the antagonistic activity of R. glutinis harvested either from the culture media of NYDB amended with chitin at 0.5% or from the culture media in which chitin was the sole carbon source (NYCB) was improved greatly compared with that without chitin. The application of R. glutinis cultivated in the culture media of the chitin-supplement (0.5%) induced higher β-1,3-glucanase activity and reduced more MDA content of strawberries compared with that R. glutinis cultivated in the NYDB. Moreover, the control efficacy of the cell-free filtrate of the chitin-supplement culture media (0.5%) and NYCB were higher than that of cell-free culture filtrates of NYDB in 2 days incubation, with the associated high level of chitinase activity.     

Sensitivity of Penicillium expansum field isolates to tebuconazole, iprodione, fludioxonil and cyprodinil and characterization of fitness parameters and patulin production

A total of 236 Penicillium expansum field isolates from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, Northern Greece were tested for their sensitivity to tebuconazole, fludioxonil, iprodione and cyprodinil. Preliminary fungitoxicity tests on the response of the isolates showed several phenotypes, distinguished according to their sensitivity to fungicides tested. The EC₅₀ values ranged from 0.64 to 5 (average = 0.98) μg/ml for iprodione, 0.9 to 7.3 (average = 2.66) μg/ml for tebuconazole, 0.008 to 1.28 (average = 0.55) μg/ml for cyprodinil and from 0.013 to 0.47 (average = 0.08) μg/ml for fludioxonil. A bimodal distribution of the EC₅₀ values of isolates with distinct sensitive and resistant populations to fludioxonil and tebuconazole were observed. In the case of cyprodinil, a much broader, hundred-fold, range of sensitivity was found, probably indicating that some isolates are relatively insensitive to cyprodinil compared to the most sensitive ones. Isolates exhibiting simultaneously reduced sensitivity to tebuconazole and fludioxonil or tebuconazole and iprodione or to tebuconazole and cyprodinil were also observed at low frequencies. A small portion of the population (7.5%) showed multiple resistance to tebuconazole, fludioxonil and iprodione. Study of fitness determining parameters showed that the resistance to tebuconazole, fludioxonil and iprodione had a significant adverse effect on mycelial growth rate and pathogenicity. Contrary to that, these fitness parameters were not affected in the isolates showing reduced sensitivity to cyprodinil. Analysis of patulin production on YES-agar growth medium and on artificially inoculated apple fruit showed that all isolates were mycotoxigenic. Most of the cyprodinil-insensitive isolates produced patulin at concentrations similar to or relatively higher (up to 1.5-fold on growth medium) than the sensitive ones. In contrast, a significant reduction (up to 98% of multiple resistant isolates) in patulin production was observed in all other phenotypes, indicating an adverse effect of fitness penalties on the mycotoxigenic ability of resistant isolates. The above mentioned data clearly show a considerable risk for the selection of P. expansum isolates resistant to fludioxonil, iprodione, tebuconazole and cyprodinil. The potential risk of increased patulin contamination of apples and their byproducts by the appearance and predominance of highly mycotoxigenic isolates of P. expansum resistant to the anilinopyrimidines is discussed.     

In vitro and in vivo antifungal activities of the essential oils of various plants against tomato grey mould disease agent Botrytis cinerea

The aim of this study was to find an alternative to synthetic fungicides currently used in the control of devastating fungal pathogen Botrytis cinerea, the causal agent of grey mould disease of tomato. Antifungal activities of essential oils obtained from aerial parts of aromatic plants, which belong to the Lamiacea family such as origanum (Origanum syriacum L. var. bevanii), lavender (Lavandula stoechas L. var. stoechas) and rosemary (Rosmarinus officinalis L.), were investigated against B. cinerea. Contact and volatile phase effects of different concentrations of the essential oils were found to inhibit the growth of B. cinerea in a dose-dependent manner. Volatile phase effects of essential oils were consistently found to be more effective on fungal growth than contact phase effect. A volatile vapour of origanum oil at 0.2 μg/ml air was found to completely inhibit the growth of B. cinerea. Complete growth inhibition of pathogen by essential oil of lavender and rosemary was, however, observed at 1.6 μg/ml air concentrations. For the determination of the contact phase effects of the tested essential oils, origanum oil at 12.8 μg/ml was found to inhibit the growth of B. cinerea completely. Essential oils of rosemary and lavender were inhibitory at relatively higher concentrations (25.6 μg/ml). Spore germination and germ tube elongation were also inhibited by the essential oils tested. Light and scanning electron microscopic (SEM) observations revealed that the essential oils cause considerable morphological degenerations of the fungal hyphae such as cytoplasmic coagulation, vacuolations, hyphal shrivelling and protoplast leakage and loss of conidiation. In vivo assays with the origanum essential oil, being the most efficient essential oil, under greenhouse conditions using susceptible tomato plants resulted in good protection against grey mould severity especially as a curative treatment. This study has demonstrated that the essential oils are potential and promising antifungal agents which could be used as biofungicide in the protection of tomato against B. cinerea.     

Efficacy of killer yeasts in the biological control of Penicillium digitatum on Tarocco orange fruits (Citrus sinensis)

Killer Saccharomyces cerevisiae and Wickerhamomyces anomalus yeast strains were tested as biocontrol agents against Penicillium digitatum, one the most important causes of postharvest decay in orange fruits.     

The effects of the combination of Pichia membranefaciens and BTH on controlling of blue mould decay caused by Penicillium expansum in peach fruit

The feasibility of 0.2 g l⁻¹ benzo-thiadiazole-7-carbothioic acid S-methyl ester (BTH) to improve the efficacy of Pichia membranefaciens in controlling postharvest blue mould decay in peach fruit was investigated. Our results showed that biocontrol activity of P. membranefaciens against blue mould caused by Penicillium expansum in peach fruit could be enhanced by addition of 0.2 g l⁻¹ BTH. The combination of P. membranefaciens and BTH resulted in a more effective control of blue mould than individual treatment of P. membranefaciens or BTH alone. The combined treatment had a synergistic effect on the induction of superoxide dismutase, catalase, ascorbate peroxidase, chitinase and β-1,3-glucanase activities, which induced stronger disease resistance in fruit than BTH or yeast alone, and resulted in a lower lesion diameter and disease incidence of blue mould decay in peaches. Furthermore, the combined treatment did not impair the quality parameters including fruit firmness and contents of total soluble solids, titratable acidity and vitamin C of peach fruit after 6 days of storage at 20 °C. These results suggested that the use of BTH may be an effective method to improve the biological activity of P. membranefaciens.     

Evaluation of Penicillium expansum isolates for aggressiveness, growth and patulin accumulation in usual and less common fruit hosts

Experiments were carried out in vivo and in vitro with four isolates of Penicillium expansum (I 1, E 11, C 28 and I 12) to evaluate their aggressiveness, growth and patulin accumulation in both usual (pears and apples) and less common hosts (apricots, peaches, strawberries and kiwifruits) of the pathogen. The 75% of isolates showed the ability to cause blue mould in all tested hosts. In particular, C 28 and I 1 were the most and the least aggressive isolates, respectively (52.9 and 10.6% infection and 20.7 and 15.4 mm lesion diameters). ‘Candonga’ strawberries and ‘Pinkcot’ apricots showed the largest lesion diameters (29.8 and 25.3 mm), followed by ‘Conference’ pears, ‘Spring Crest’ peaches and ‘Abate Fetel’ pears. With the exception of ‘Candonga’ strawberries, the formation of colonies and mycelial growth of P. expansum isolates on fruit puree agar media (PAMs) was stimulated in comparison to a standard growth medium (malt extract agar, MEA). Two of the most aggressive isolates in our assays (I 12 and C 28) showed the greatest accumulation of patulin both in vitro and in vivo, while the least aggressive isolate (I 1) produced patulin only in a few growth media and cvs. Patulin concentration on fruit PAMs was higher than patulin detected in infected fruit tissues. Apple PAMs were the more favorable substrates for patulin accumulation in vitro (maximum concentration 173.1 and 74.1 μg/mL in ‘Pink Lady and ‘Golden Delicious’ PAMs, respectively) and ‘Pink Lady’ apples inoculated with the isolate E 11 showed the greatest accumulation of patulin in the whole in vivo assay (33.9 μg/mL). However, infected tissue of cv Golden Delicious showed lower average accumulation of patulin (1.7 μg/mL) than that of cv Pink Lady (19.1 μg/mL), and no significant differences in patulin concentrations were found among ‘Golden Delicious’ apples and tested cvs of pears, kiwifruits and strawberries. Peaches were highly susceptible to patulin accumulation, showing average concentrations of 27.4 and 18.6 μg/mL in vitro and in vivo, respectively. Apricots were also consistently positive for patulin accumulation, both in vitro (average values of 20.1 μg/mL) and in vivo (average values of 9.4 μg/mL). Our study showed the potential of some less common hosts of P. expansum (in particular peaches and apricots) to support patulin production, indicating that a steady monitoring of patulin contamination should be carried out in fruit substrates other than apples and pears.     

Identification and characterization of a chitinase of Stenotrophomonas maltophilia, a bacterium that is antagonistic towards fungal phytopathogens

A rhizosphere strain of Stenotrophomonas maltophilia strain MUJ that is strongly antagonistic towards fungal phytopathogens secretes to the culture medium a single form of active chitinolytic enzyme belonging to family 18 of glycosyl hydrolases. The chitinase was purified by a two-stage procedure embracing fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme determined by SDS-PAGE was approximately 52 kDa. The enzyme demonstrated highest activity at 45°C and pH 6.8. The enzymatic protein showed considerable thermal stability during 2 h incubation at 45°C. The activity of the enzyme was strongly inhibited in the presence of Hg²⁺ and Cu²⁺. By applying mass spectrometry analysis, the peptides derived from the purified chitinase were assigned to amino acid sequences of the type ChiA chitinases synthesized by Stenotrophomonas bacteria. The purified enzyme inhibited the growth of fungal phytopathogens belonging to the genera Fusarium, Rhizoctonia and Alternaria.     

Impact of noble rot on the aroma precursor of 3-sulfanylhexanol content in Vitis vinifera L. cv Sauvignon blanc and Semillon grape juice

The most famous sweet wine in France is made from botrytised grapes in the Sauternes region. Its distinctive, complex aromas are undoubtedly due to noble rot as a result of Botrytis cinerea development on perfectly ripe Sauvignon blanc and Semillon grapes. The contribution of volatile thiols in botrytised wine aroma was recently reported. The objective of this work was to investigate the impact of B. cinerea development on the aromatic potential of grape juice. The level of S-3-(hexan-1-ol)cysteine (P-3SH), the cysteinylated precursor of the most abundant volatile thiol in wine (3-sulfanylhexanol, 3SH), was determined during grape botrytisation and several grape overripening processes. Production was considerably amplified when B. cinerea had developed on the grapes in both on- and off-vine experiments. The P-3SH levels were increased about 100-fold between the healthy and botrytised stages in one week. We demonstrated that the grape metabolism produced aroma precursor under post-harvest conditions. When grapes were left to overrippen, the vine also produced P-3SH, but levels increased more moderately (about 10-fold). Moreover, a determination of P-3SH distribution demonstrated that B. cinerea was not directly responsible for precursor formation but probably stimulated the grape metabolic pathway involved in this formation.     

A laboratory assessment of the potential of different strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in stored cowpea

Twelve indigenous and exotic isolates of Beauveria bassiana and Metarhizium anisopliae were evaluated for their virulence and their ability to suppress populations of Callosobruchus maculatus in stored cowpea. LT₅₀ values ranged from 3.11 to 6.12 days following immersion in aqueous suspensions containing 1×10⁸ conidia ml⁻¹. Indigenous isolates that had been recovered from C. maculatus were more virulent in laboratory bioassays than exotic isolates from other insects. The two isolates with the shortest LT₅₀ values were compared in dose-response assays by immersion and by exposure to cowpea grains treated with dry conidia. In both assays B. bassiana 0362 was consistently more virulent than M. anisopliae 0351. By immersion, LC₅₀ values on day 6 post-treatment were 9.10×10⁴ and 7.10×10⁵ conidia ml⁻¹ for B. bassiana 0362 and M. anisopliae 0351, respectively. Exposure to treated grains gave LC₅₀ values of 1.15×10⁷ and 4.44×10⁷ conidia g⁻¹ grain for B. bassiana 0362 and M. anisopliae 0351, respectively. In 1 kg batches of cowpea stocked with 50 adult C. maculatus, B. bassiana 0362 at both 1×10⁷ and 1×10⁸ conidia g⁻¹ grain led to significant adult mortality and reduced F₁ emergence relative to untreated populations. At 1×10⁸ conidia g⁻¹ the effect of the fungus persisted into the F₁ generation. The net reproductive rates, R₀, measured 26 days after insects were released were 5.16 and 7.32 for the high and low doses compared to 9.52 for the untreated control.     

Anti- and pro-oxidant activity of Cichorium genus vegetables and effect of thermal treatment in biological systems

The antiradical activity of water soluble components in six vegetables belonging to the Cichorium genus, i.e., three cultivars of red intybus species var. silvestre (Treviso, Chioggia, Verona red chicories), a white intybus species var. foliosum (Belgian chicory), and two vegetables of the endivia species var. latifolium (escarole chicory) and var. crispum (“crispa” chicory), were studied using two biological systems consisting of: (1) microsome membrane rat hepatocyties in which oxidative damage was induced by CCl₄; (2) gram-positive bacterium, Staphylococcus aureus cultures, subjected to damage with cumene hydroperoxyde. The obtained results show that in both systems the red vegetables possess the strongest antioxidant properties and contain different antioxidant compounds whether at a low or high molecular weight, but only those of high molecular-weight (MW > 3500 Da) are able to act as antioxidants in all the used systems. The lower MW fraction (MW < 3500 Da) showed itself to be pro-oxidant in the microsome system. The effects of thermal treatments such as boiling, freezing and freeze-drying were also investigated.     

Citrus phenylpropanoids and defence against pathogens. Part II: Gene expression and metabolite accumulation in the response of fruits to Penicillium digitatum infection

The effect of infection of Citrus sinensis (var. Navelina) fruits with Penicillium digitatum was studied at gene expression and metabolite levels. In this study, expression of genes involved in the phenylpropanoid pathway was studied in the flavedo (outer coloured part of the peel) and albedo (inner white part) in response to pathogen infection. Results of the time-course experiment showed that maximal expression of 10 out of 17 phenylpropanoid genes analysed occurred at 48 h post-inoculation, when decay symptoms started to appear, and mRNA levels either kept constant or decreased after 72 h post-inoculation. To further investigate the putative involvement of the phenylpropanoid pathway in the defence of citrus fruit, changes in the metabolic profile of both tissues infected with P. digitatum was studied by means of HPLC-PDA-FD. Metabolite accumulation levels along the time course suggest that flavanones, flavones, polymethoxylated flavones and scoparone are induced in citrus fruit in response to P. digitatum infection, although with different trends depending on the tissue.     

Cloning and characterization of a novel phytase from wastewater treatment yeast Hansenula fabianii J640 and expression in Pichia pastoris

Phosphohydrolysis of organic phosphorus compounds by acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2) is an important method for efficient removal of phosphorus from high concentration organic wastewater. Another important method is supplementation of animal feed with phytase (EC 3.1.3.8 and EC 3.1.3.26), which improves the availability of phytate-phosphates (phosphate that are hydrolyzed by phytases), making it possible to add less phosphate to animal feed and resulting in the excretion of less phosphorus by the animals. In the present study, we purified a novel phytase from the wastewater treatment yeast Hansenula fabianii J640 (Hfphytase), cloned the 1456 bp open reading frame (ORF) encoding Hfphytase, and characterized Hfphytase. The molecular weight of Hfphytase after deglycosylation by PNGaseF was 49 kDa. The optimal pH and temperature for enzyme activity were 4.5 and 50 °C, respectively. Hfphytase exhibits 40% identity with Debaryomyces castellii phytase, 37% identity with Aspergillus niger PhyB, and 34% identity with Saccharomyces cerevisiae Pho5p. Recombinant Hfphytase was transformed and expressed in Pichia pastoris. The yield was 23 g/l by jar fermenter cultivation. The marked phosphohydrolysis activity exhibited by Hfphytase on six substrates (pNP-P, sodium phytate, glucose-1 phosphate, glucose-6 phosphate, α-glycerophosphate and β-glycerophosphate) indicated that it is a non-specific acid phosphatase.     

Cloning of Aspergillus oryzae Aovps5 gene,homologous to vacuolar protein sorting associated gene VPS5 and construction of the disruptant

Aovps5 gene was isolated from Aspergillus oryzae as a homologue to S. cerevisiae VPS5 gene which encodes a polypeptide consisting of 451 amino acids that is nearly 32% homologous to Vps5p. Three Aovps5 gene disruptants were generated and they showed higher activity of tripeptidyl peptidase, which is mainly detected in vacuoles, in their culture medium. Higher amount of nitrogenous constituent was found in the filtrate of wheat gluten degraded by addition of culture medium of these disruptants than that of wild type strain. These results suggest that disruption of Aovps5 may contribute to production of fermented foods.     

Effects of cold storage, rearing temperature, parasitoid age and irradiation on the performance of Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae)

In this study, the effects of cold storage, rearing temperature, parasitoid age, and irradiation on the performance of the egg parasitoid Trichogramma evanescens were investigated. Pupae of T. evanescens can be stored at 4 °C for up to 3 weeks without much loss of performance. The longevity and walking speed of adults emerging from chilled pupae significantly decreased after longer storage periods. The F₁ generation of adults which emerged from pupae stored up to 3 weeks was able to parasitize as well as the control. The parasitization rate was similar at 24, 27, and 30 °C, but significantly decreased at 33 and 36 °C. Although T. evanescens developed to the pupal stage at 36 °C, no adult emergence was observed at this temperature. Developmental periods were longer at 24 °C than at higher temperatures. The optimum age for T. evanescens to successfully parasitize host eggs ranged from 24 to 90 h. The parasitization frequency of the 56-78 h aged females was higher than for the other age groups. The daily egg laying pattern of female T. evanescens adults was similar when they were reared on Ephestia kuehniella or Plodia interpunctella eggs. Gamma- or ultraviolet-irradiated and unirradiated host eggs were equally preferred by adult females.     

Preparation,preliminary characterization and immunostimulatory activity of polysaccharide fractions from the peduncles of Hovenia dulcis

The peduncles of Hovenia dulcis, containing abundant nutrients and having a taste like a combination of raisin, clove, cinnamon and sugar, have been consumed as fruits and used as traditional herbal medicine for a long time in China. Up to date, little information is available about the polysaccharides from peduncles of H. dulcis (HDPS) and their potential bioactivity. In this study, three purified fractions were prepared by sequential purification of crude HDPS through ion-exchange chromatography and gel-filtration chromatography. The three fractions of HDPS-1, HDPS-2 and HDPS-3 were found to be homogeneous heteropolysaccharides mainly composed of rhamnose, arabinose, galactose and galacturonic acid with an average molecular weight of 235, 70 and 53 kDa, respectively. HDPS-3 was quite different from HDPS-1 and HDPS-2, as it contained much higher content of galacturonic acid (40.5%). In vitro immunostimulatory activity evaluation revealed that all the three fractions could significantly stimulate the proliferation of splenocytes and enhance phagocytosis, nitric oxide production and acid phosphatase activity of peritoneal macrophages, which suggested that HDPS had a potent immunostimulatory activity and could be explored as a potential natural immunomodulatory agent.     

Fecundity and ability of the parasitoid Lariophagus distinguendus (Hymenoptera: Pteromalidae) to find larvae of the granary weevil Sitophilus granarius (Coleoptera: Curculionidae) in bulk grain

Fecundity and host finding behavior in the storage environment were examined for the parasitoid Lariophagus distinguendus (Förster), a potential agent for biological control of stored-product pest beetles. Larvae of the granary weevil, Sitophilus granarius (L.), in wheat were used as hosts. The fecundity varied considerably among strains of different geographical origin, from 1.3±2.4 to 92.5±23.6 offspring per female parasitoid. Host finding ability was examined in a small acrylic cylinder (about 42 kg wheat), a silo-bin (20.5 t wheat grain), and the flat storage grain bin in a commercial facility (3000 t rye). Experiments in the cylinder and the silo-bin revealed that the number of hosts found by the parasitoids decreases with increasing depth. Moreover, parasitoids were more likely to find hosts in the cylinder than under field storage conditions in a silo-bin. This indicates that the density of parasitoids per unit volume of the grain might be an important factor in determining how deep they penetrate a grain layer. In the silo-bin and the commercial grain store, parasitoids were able to find and parasitize hosts located up to 4 m vertically and horizontally from the release point. The significance of these results with respect to the suitability of L. distinguendus for biological control of the granary weevil is discussed.