M. Lodovica Gullino

Uses and challenges of novel compounds for plant disease control

Abstract Fungicide research has produced a diverse range of products with novel modes of action during the last few years. These new classes of fungicides which include anilinopyrimidines, phenoxyquinolines, oxazolidinediones, spiroketalamines, scytalone dehydratase inhibitors, phenylpyrroles, strobilurins, and activators of systemic acquired resistance are expected to have a significant impact on disease control in the next decade. The most recent results in fungicide discovery originated from different approaches: traditional empirical screening of synthetic chemicals; traditional screening of natural products; biochemically directed screening of chemicals to discover; non-conventional agents stimulating plant defense. This review considers the different classes of recently developed fungicides, particularly focusing on their mode of action, resistance risk and impact on disease control.

Biocontrol capability of Metschnikowia pulcherrima against Botrytis postharvest rot of apple

Abstract The biocontrol capability of two isolates of the yeast Metschnikowia pulcherrima against Botrytis postharvest rot of apple was studied in vitro and on apples under different storage conditions. The biocontrol activity of M. pulcherrima 2.33 or 4.4 can be reduced or totally suppressed by the addition of several nitrates. The addition of 100 g l−1 fructose inhibited the pathogen per se, while leaving unaltered the antagonistic capability of M. pulcherrima. These results support the hypothesis that competition for nutrients plays a major role in the biocontrol capability of M. pulcherrima against Botrytis postharvest rot of apple. However, both yeasts strongly inhibited the growth and spore germination of B. cinerea in vitro even under non-restrictive nutrient conditions. There was no evidence for the involvement of diffusible toxic metabolites in the biocontrol efficacy. The antagonistic activity of M. pulcherrima was dependent on the concentration of the antagonist. At 107–108 cells ml−1, 2.33 was most effective when applied at least 6 h before the pathogen, while 4.4 provided consistent results when co-inoculated with B. cinerea or applied 1 h later. Rapid colonisation of fresh apple fruit wounds was observed at room temperature during the first 24–48 h, and then the populations stabilised for the remaining storage period. On apple wounds kept at 4°C, the increase in population density of M. pulcherrima was lower, but continued over 96–128 h after application of the antagonists until it reached the same saturation level observed on apples stored at room temperature.

Identification of Fusarium oxysporum f. sp. basilici Isolated from Soil, Basil Seed, and Plants by RAPD Analysis

Fifty-two isolates of Fusarium oxysporum, obtained from infected basil plants, seed, flower residues, and soil from different growing areas in Italy and Israel, were analyzed by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), coupled to a DNA extraction protocol from colonies grown on Fusarium-selective medium. In a pathogenicity assay, 35 isolates caused 32 to 92% disease on seedlings of the highly susceptible basil cultivar Fine verde, while 17 isolates were nonpathogenic on basil. Thirty of the F. oxysporum f. sp. basilici isolates obtained from soil or wilted plants gave identical amplification patterns using 31 different random primers. All tested primers allowed clear differentiation of F. oxysporum f. sp. basilici from representatives of other formae speciales and from nonpathogenic strains of F. oxysporum. RAPD profiles obtained from DNA of isolates extracted directly from cultures grown on Fusarium selective medium were identical to those obtained from DNA extracted from lyophilized mycelia.

Detection of enzymatic activity and partial sequence of a chitinase gene in Metschnikowia pulcherrima strain MACH1 used as post-harvest biocontrol agent

Two antagonistic yeast strains Metschnikowia pulcherrima MACH1 and Rhodotorula sp. PW34 were tested for their efficacy against Botrytis cinerea in vitro and in vivo on apples. Metschnikowia pulcherrima strain MACH1 showed higher inhibition of B. cinerea compared to the strain PW34 in vitro on potato dextrose broth. Further, yeast strain MACH1 showed higher efficacy in reducing grey mould on apples compared to PW34 and the untreated control. In addition, partially purified extracellular proteins from strain MACH1 showed an inhibition to B. cinerea in vitro. The antagonistic yeast strains were tested for their efficacy to produce chitinases in different liquid media, including apple juice, amended with or without cell wall preparations (CWP) of B. cinerea. The study showed a higher production of chitinases from M. pulcherrima strain MACH1 when compared to PW34. Interestingly, the strain MACH1 secreted higher chitinases in the presence of cell wall fractions of B. cinerea. For this reason, the chitinase gene of strain MACH1 was amplified using PCR reactions and the nucleotide sequence data showed high homology to chitinases of other yeast strains. The results of the current study show that M. pulcherrima strain MACH1 has the ability to secrete chitinases in different liquid media including apple juice, and the enzyme could be involved in the post-harvest biological control of B. cinerea.

Application of selected antagonistic strains against Phytophthora cryptogea on gerbera in closed soilless systems with disinfection by slow sand filtration

Abstract In two separate trials during 2000–2002, the efficacy of slow sand filtration and UV treatment in eliminating Phytophthora cryptogea propagules, artificially added in the recirculating nutrient solution, was evaluated with gerbera plants grown in closed soilless systems. A slow sand filtration technique was tested both alone and in combination with different antagonistic strains belonging to Fusarium spp. and Trichoderma spp., isolated from gerbera rhizosphere and applied into the soilless system. The dynamics of these fungi in the recirculating nutrient solution and in the sand filter was also investigated by plate counts on selective media. Slow sand filtration and UV treatment were both effective in reducing P. cryptogea root rot. However, slow sand filtration may be a more feasible disinfection method than UV because of lower costs of installation and maintenance and for its adaptability to a wide range of production systems. Moreover, this disinfection technique can be successfully combine with the application of antagonistic microorganisms.

Limited vegetative compatibility following intra- and interspecific protoplast fusion inTrichoderma

Abstract A variety of auxotrophic mutants were prepared from several species and strains of Trichoderma by nitrosoguanidine (NNG) mutagenesis. Effective protoplasting from hyphae was achieved with the commercial enzyme preparation Novozym 234; however, pretreatment with 2-deoxy- d -glucose was required for several strains. Aggregation of protoplasts and subsequent fusion were monitored directly by complementary fluorescent staining and were effectively induced by polyethylene glycol and calcium regardless of the Trichoderma species, strains, or auxotrophs being fused. In all cases, about 10 6 viable colony-forming units (CFUs) were formed from about 2 × 10 8 protoplasts. However, subsequent recovery of somatic hybrid colonies was dramatically lower for interstrain fusions than for intrastrain (between two auxotrophs derived from one strain) fusions. Following intrastrain fusions, 2 to 10 × 10 −2 of the viable CFUs grew under selective conditions regardless of the auxotrophs involved, indicating that induced heterofusions were frequent and nutritional complementation was functional. In interstrain fusions, however, only about 1 to 20 × 10 −5 of the viable CFUs produced colonies under selective conditions, indicating a low level of postfusion compatibility. Restricted growth of these somatic hybrid colonies, which were not heterokaryotic, appears to result from fusion of heterologous protoplasts and vegetative incompatibility. No vegetatively compatible pairs of strains were resolved; all inter- and intraspecific protoplast fusions exhibited similarly limited compatibility. Limited compatibility may reduce the likelihood of parasexual recombination but does not preclude the possibility of genetic manipulation of Trichoderma strains by protoplast fusion.

Effect of antagonistic Fusarium spp. and of different commercial biofungicide formulations on Fusarium wilt of basil (Ocimum basilicum L.)

Abstract The ability of antagonistic Fusarium spp. to control Fusarium wilt of basil caused by F. oxysporum f. sp. basilici was tested under glasshouse conditions from 1994 to 1996. Fusarium oxysporum antagonistic strain 251/2, a protoplast fusion-derived hybrid coded FI-11 and F. moniliforme strains TF4 and TF4 RB were able to decrease ( P = 0.05) the incidence of Fusarium wilt significantly. Biocontrol was consistent, especially when the antagonists were applied by mixing a talc preparation of propagules in the substrate (10 5 CFU/ml of substrate) 1–2 weeks before sowing or at sowing. Seed coating with the biocontrol strains (10 8 CFU/g of seed) did not generally provide sufficient disease containment, whereas the combination of substrate treatment and seed coating did not improve wilt control in comparison with substrate treatment alone. Two commercial formulations, based on antagonistic F. oxysporum strains and one containing the antagonistic strain K61 of Streptomyces griseoviridis , were generally not sufficiently effective at the tested dosages.

Recent developments in management of plant diseases

Introduction U. Gisi, I. Chet, M.L. Gullino.- List of contributors.- Part I. Concepts in chemical control.- 1. The Challenges of Chemical Control of Plant Diseases A. Leadbeater, U. Gisi.- 2. Novel Tools to Identify the Mode of Action of Fungicides as exemplified with Fluopicolide V. Toquin et al.- 3. QoI fungicide resistance: current status and the problems associated with DNA-based monitoring H. Ishii.- 4. Regulatory Aspects in Chemical Control of Fungal Diseases: Impact on Efficient Plant Production G. F. Backhaus.- Part II. Concepts in biological control of plant pathogens.- 5. The roles of cyclic lipopeptides in the biocontrol activity of Bacillus Subtilis M. Ongena et al.- 6. Biocontrol of Plant Pathogens and Plant Growth Promotion by Bacillus B. B. McSpadden Gardener.- 7. Importance of multitrophic interactions for successful biocontrol of plant parasitic nematodes with Paecilomyces lilacinus strain 251 S. Kiewnick.- 8. Interactions between Clonostachys rosea f. catenulata, Fusarium oxysporum and cucumber roots leading to biological control of Fusarium root and stem rot S. Chatterton, Z. K. Punja.- 9. Screening of biocontrol agents for control of foliar diseases J. Kohl.- 10. Quorum sensing as a target for novel biocontrol strategies directed at Pectobacterium A. Cirou et al.- 11. The status of biological control of plant diseases in soilless cultivation J. Postma.- 12. Rebeca - EU Policy Support Action to review Regulation of Biological Control Agents R.-U. Ehlers.- 13. Fungal Disease management in organic apple orchards: epidemiological aspects and management approaches I.J. Holb.- Part III. Natural compounds.- 14. Exploitation of natural compounds in eco-friendly management of plant pests N. K. Dubey et al.- 15. Regulation and functional analysis of bioprotective metabolite genes from the grass symbiont Epichloe Festucae B. Scott et al.- Part IV. Soilborne plant diseases and their control.- 16. IPM for soilborne disease management for vegetable and strawberry crops in SE USA F. J. Louws.- 17. Integrated Control of Soilborne Pathogens of Wheat T. C. Paulitz et al.- 18. Challenges in Controlling Verticillium Wilt by the Use of Nonchemical Methods G. Lazarovits, K. Subbarao.- 19. Soil solarisation - 30 years on: what lessons have been learned? J. Katan, A. Gamliel.- 20. Back to the future: total system management (organic, sustainable) D. O. Chellemi.- 21. Global phaseout of methyl bromide under the Montreal Protocol: Implications for bioprotection, biosecurity and the ozone layer I. Porter et al.- 22. Accelerated degradation of soil fumigants: occurrence and agricultural consequences A. Gamliel, S. Triky-Dotan.- 23. Behavior of fumigants in soil W. Ntow, H. Ajwa.- Part V. Plant breeding and resistance strategies.- 24. Worldwide monitoring systems: the need of public and private collaboration R. D. Magarey et al.- 25. Identification of new sources of resistance to soybean rust S. Li.

Chemical and Non Chemical Seed Dressing for Leafy Vegetable Crops

Using healthy seeds is a prerequisite in any cropping systems, in order to reduce the further adoption of other disease management strategies in the field during the cultivation. Since seeds are often contaminated, also if at a very low level, by seed-borne pathogens, seed dressing is considered an important method for disease prevention. This is particularly true in the case of seeds of vegetable crops, which very often carry the inoculum of important pathogens. It is well proven with many pathosystems, that a very low percent of infected seeds is able to cause severe losses under greenhouse and field conditions. Seed dressing with chemicals was largely adopted for many decades, because of the availability of effective fungicides, at relatively low cost and the easiness of the treatment. However, recent restrictions in the registration and use of chemicals, stimulated the re-evaluation of old non chemical methods as well as the development of new ones.

Sensitivity to Anilinopyrimidines and Phenylpyrroles in "Botrytis cinerea" in North-Italian Vineyards.

Several commercial vineyards, located in Piedmont (Northern Italy), were monitored in order to evaluate the sensitivity of Botrytis cinerea Pers., the causal agent of grey mould, to five classes of botryticides: benzimidazoles, dicarboximides, phenylcarbamates, anilinopyrimidines and phenylpyrroles. Strains of B. cinerea resistant to anilinopyrimidines were easily detected, particularly in 1999, a year characterized by high disease pressure, even in vineyards not sprayed with that class of fungicides. Fludioxonil-resistance, on the contrary, was not detected. Resistance to benzimidazoles and dicarboximides was at previous observed levels. For the first time, resistance to phenylcarbamates was detected in the field. Strains of B. cinerea showing multiple resistance to benzimidazoles, dicarboximides and anilinopyrimidines and maintaining a good level of virulence, as shown by tests carried out on wounded apples, are present in Italian vineyards. Strategies in the use of the botryticides are discussed, in order to avoid a loss of disease control.