Gennaro Cristinzio

Isolation, spectroscopy and selective phytotoxic effects of polyphenols from vegetable waste waters

Abstract Catechol, 4-methylcatechol, tyrosol and hydroxytyrosol were isolated and characterized as the main polyphenols from olive oil mill waste waters. In addition, the corresponding acetates were prepared. In phytotoxicity assays carried out on tomato (Lycopersicon esculentum) and vegetable marrow (Cucurbita pepo) plants, the compounds were selectively toxic, except for 4-methylcatechol and its acetate. The vegetation waters remained phytotoxic even after total extraction of the polyphenols, suggesting that other chemical products contribute to the overall phytotoxicity.

Detection and identification of Phytophthora species in southern Italy by RFLP and sequence analysis of PCR-amplified nuclear ribosomal DNA

In four neighbouring regions of southern Italy, Basilicata, Campania, Apulia and Calabria, pepper and zucchini plants showing Phytophthora blight symptoms, tomato plants with either late blight or buckeye rot symptoms, plants of strawberry showing crown rot symptoms and declining clementine trees with root and fruit rot were examined for Phytophthora infections by means of polymerase chain reaction (PCR) assays, using primers directed to nuclear ribosomal DNA (rDNA) repeat sequences. All diseased plants and trees examined tested positive. The detected fungal-like organisms were differentiated and characterized on the basis of primer specificity as well as through extensive restriction fragment length polymorphism (RFLP) and sequence analysis of PCR-amplified rDNA. Phytophthora capsici was identified in diseased pepper and zucchini plants, P. infestans was identified in tomato with late blight symptoms whereas buckeye rot-affected tomatoes and diseased strawberry plants proved to be infected by P. nicotianae and P. cactorum, respectively. Declining clementine trees were infected with P. citrophthora and P. nicotianae in about the same proportion. Also, thirty-one pure culture-maintained isolates of Phytophthora which had previously been identified in southern Italy by traditional methods but were never examined molecularly, were examined by RFLP and sequence analysis of PCR-amplified nuclear rDNA. Among these, an isolate from gerbera which had previously been identified by traditional methods only at genus level, was assigned to P. tentaculata. For the remaining pure culture-maintained isolates examined, the molecular identification data obtained corresponded with those delineated by traditional methods. Most of the diseases examined were already known to occur in southern Italy but the pathogens were molecularly detected and fully characterized at nuclear rDNA repeat level only from other geographic areas, very often outside Italy. A new disease to southern Italy was the Phytophthora blight of zucchini. This is also the first report on the presence and molecular identification of P. tentaculata from Italy.

Direct and mediated effects onBactrocera oleae (Gmelin) (Diptera; Tephritidae) of natural polyphenols and some of related synthetic compounds: Structure-activity relationships

Among the main polyphenols occurring in olive oil vegetation waters (VW), catechol showed the most deterrent action on the oviposition ofBactrocera oleae (Gmelin); 4-methylcatechol was less active, whereas hydroxytyrosol and tyrosol were inactive. In contrast, synthetico-quinone was found to be stimulant at 7.5 × 10−2 M. Two other synthetic derivatives of catechol, diacetylcatechol and guaiacol, were also deterrent, suggesting these compounds undergo a biochemical transformation into catechol by means of the bacterial symbionts ofB. oleae. VW and their phenolic extracts showed deterrence only when highly concentrated, while natural olive juice was strongly deterrent. Experiments carried out to evaluate the effect of olive juice and catechol on the fecundity ofB. oleae showed that they strongly reduce this function. Moreover, the possible utilization of VW and their bioactive polyphenols in protection of olives againstBatrocera oleae is discussed.

Flufuran, an antifungal 3,5-disubstituted furan produced by Aspergillus flavus link.

A 3,5-disubstituted furan, named flufuran, was isolated from a culture filtrate of a strain of Aspergillus flavus obtained from a chestnut compost created in the same orchard. Flufuran was identified by spectroscopic methods, and its structure was confirmed through the preparation of some key derivatives, also used to test the antifungal activity. At a concentration of 0.2 mg/ml, assayed against three Phytophthora species, pathogenic of some forest and agrarian plants, flufuran and especially its acetyl derivative showed significant antifungal activity. Although flufuran appears to be identical to a fungal metabolite isolated previously from some Polyporus spp., its interesting antifungal activity has never been reported before.

Biosorption of pentachlorophenol by Anthracophyllum discolor in the form of live fungal pellets

Pentachlorophenol (PCP) is an extremely dangerous pollutant for every ecosystem. In this study we have detected how PCP concentration and pH levels can influence PCP adsorption by Anthracophyllum discolor in the form of live fungal pellets. PCP adsorption was evaluated after 24 hours in KCl 0.1 M electrolyte solution with initial PCP concentrations of 5 and 10 mg L (-1) and with pH values between 4 and 9 (at intervals of 0.5). Fourier Transform Infrared Spectroscopy (FTIR) was used to identify functional groups of fungal biomass that can interact with PCP. The amount of PCP that was adsorbed by A. discolor was >80% at pH values between 5 and 5.5, whatever the concentration tested. PCP adsorption significantly decreased in liquid medium of pH > 6.0. FTIR results showed that amides, alkanes, carboxylates, carboxyl and hydroxyl groups may be important to the PCP adsorption for pellets of A. discolor. Live fungal pellets of A. discolor may be used as a natural biosorbent for liquid solutions contaminated by PCP.

First report of Phytophthora insolita and P. inflata on rhododendron in Ohio.

During August 2003, we conducted a statewide survey of rhododendrons to determine if Phytophthora ramorum was present in Ohio ornamental nurseries. In total, 240 samples were randomly collected in 12 nurseries throughout Ohio from rhododendrons showing foliar necrotic lesions and twig dieback symptoms. The samples yielded 51 Phytophthora spp. isolates on PARP-V8 agar. The internal transcribed spacer (ITS) region of all isolates was amplified using the universal primers ITS1 and ITS4 and was sequenced. Consensus sequences from sense and antisense were then blasted against the GenBank database, allowing for the identification to species of ˜80% of all isolates. These identifications, and the ˜20% unknowns, were confirmed using blind morphological tests on the basis of the following parameters: colony morphology; shape and dimensions of sporangia and type of papillae; dimensions of oogonia and oospores; type and position of antheridia; presence or absence of chlamydospores; presence or absence and morphology of hyphal swellings; and growth rate at 35°C according to the Revisited Tabular Key of the species of Phytophthora (1). No P. ramorum was detected among the isolates; however, P. cactorum, P. citricola, P. citrophthora, and P. nicotianae were detected. We also found two occurrences of P. inflata Caros & Tucker and one of P. insolita Ann & Ko. (P. inflata: e-value ≤e-179, identities ≥95%; P. insolita: e-value = 0.0; identities = 95%.) P. inflata was isolated from two tissue types, a dead twig and a necrotic leaf tip. P. insolita was isolated from a necrotic leaf tip. Identity of the two species was confirmed morphologically using the parameters listed above as well as the following measurements (N = 40; all in μm) (1): P. inflata - sporangia: 40 × 24 ([24 to 68] × [18 to 34]); oogonia: 34.6 (28 to 40); oospores: 30.8 (25 to 38); P. insolita - sporangia: 42 × 28 ([34 to 56] × [22 to 38]); oogonia: 32 (26 to 36); oospores: 26 (22 to 30). Kochs postulates were satisfied by inoculating two rhododendron plants (cvs. PJM and Nova Zembla) with the putative pathogens. On each plant, each of three leaves was pierced with a dissecting needle and was inoculated by placing a 0.5-cm-diameter plug of mycelium that was taken from the margin of a colony actively growing on PARP-V8 agar on the wound. The inoculum was retained using clear adhesive tape. A similar procedure was used for twigs. Controls consisted of inoculations with sterile PARP-V8 agar medium. Both cultures of P. inflata and P. insolita produced necrotic lesions in all inoculations on both tissue types within 1 week, and they were reisolated from the margins of lesions on PARP-V8. The lesion margin was at least 2 cm away from the inoculum plug in leaf inoculations and several centimeters in twig inoculations. To our knowledge, this is the first report of P. inflata and P. insolita occurring on rhododendron and the first time P. insolita has been reported outside of Southeast Asia where it has been recovered only from soil. Reference: (1) D. J. Stamps et al. Mycol. Pap. No. 162. CAB Int. Mycol. Inst. Wallingford, UK, 1990.

Potato R1 resistance gene confers resistance against Phytophthora infestans in transgenic tomato plants

Tomato is challenged by several pathogens which cause loss of production. One such pathogen is the oomycete Phytophthora infestans which is able to attack all the aerial parts of the plant. Although a wide range of resistance sources are available, genetic control of this disease is not yet successful. Pyramiding R-genes through genetic transformation could be a straightforward way to produce tomato and potato lines carrying durable resistance to P. infestans. In this work the R1 potato gene was transferred into tomato lines. The tomato transgenic lines were analyzed by using q-RT-PCR and progeny segregation to determine the gene copy number. To test the hypothesis that R1 represents a specifically regulated R-gene, transgenic tomato plants were inoculated with P. infestans isolate 88133 and IPO. All the plants containing the R1 gene were resistant to the late blight isolate IPO-0 and susceptible to isolate 88133. These results provide evidence for specific activation of the R1 gene during pathogen challenge. Furthermore, evidence for enhancement of PR-1 gene expression during P. infestans resistance response was obtained.

Resistance traits and AFLP characterization of diploid primitive tuber-bearing potatoes

Worldwide, a variety of pathogens negatively affect potato production, resulting in an estimated 22% annual yield reduction. Wild Solanum species represent a unique gene pool where all the traits necessary to improve the cultivated potato can be found. Therefore, breeding efforts for improved disease resistance and research aimed at characterizing wild germplasm have been extensively made. In this paper, sources of resistance to Phytophthorainfestans, Erwinia carotovora subsp. carotovora, Fusarium solani and Globodera spp. have been investigated in several clones of two Solanum species originating from Central Mexico (S. bulbocastanum and S. cardiophyllum). Interestingly, we found sources of combined resistance to late blight and bacterial soft rot. This is an important finding considering that the development of resistant potato varieties has been hindered by the scarcity of resistant germplasm. In addition, we explored molecular differences within and between the two species generating AFLP fingerprints. By means of six primer pair combinations, we found 13 and 16 putative species-specific AFLP markers for S. bulbocastanum and S. cardiophyllum, respectively, and a bounty of markers useful for mapping, MAS, and cloning purposes. The phenotypic and molecular information associated to S. bulbocastanum and S. cardiophyllum for designing strategies of assisted selection are discussed.

Syringicin, a new α-elicitin from an isolate of Phytophthora syringae, pathogenic to citrus fruit

Syringicin (Syr), a new acidic α-elicitin, has been isolated from culture filtrates of Phytophthora syringae, which is the causal agent of citrus fruit rot. Its molecular weight (10194.6±0.2) determined by ES-MS is very similar to that calculated using the mean isotopic composition and considering the occurrence of disulfide bridges. The primary structure was obtained by the combined use of Edmann degradation with MALDI-MS and by comparison with already known a-elicitins. Syr induces a hypersensitive response and electrolyte leakage in tobacco. These are characteristic elicitor properties of the group and in agreement with the molecular mechanism recently proposed for this kind of protein. Finally, its possible applications in biological agriculture and biomedicine are briefly discussed.

Oleuropein and Other Polyphenols from Olive (Olea europea) for Protecting the Plant Against Pseudomonas syringae subsp. savastanoi

p ]Among the main polyphenols isolated from waste waters of olive (Olea europea) mill, catechol, 4-methylcatechol and hydroxytyrosol were toxic for Pseudomonas syringae subsp. savastanoi, the causai agent of olive knot disease. In particular the most toxic was 4-methylcatechol, Among some synthetic derivatives of catechol, as o-quinone, guaiachol, veratrole and diacetylcatechol, which were tested on P. savastanoi and on Corynebacterium michiganense (Gram+), o-quinone was strongly toxic for both bacteria, whereas veratroie and diacetylcatechol were toxic only for P. savastanoi. From fruits and leaves of olive was isolated oieuropein, a well known phenolic glucoside, from which was prepared its agtycone, by using a commercial β-giucosidase and hydroxytyrosol by chemical hydroiysis. The oieuropein was significantly toxic on P. savastanoi at a concentration of 10-3 and 10-4 M, while its aglycone was toxic in the range between 10-2 and 10-4 M and hydroxytyrosot at 10-2 and 10-3 M.