Maria Lodovica Gullino

State of the art and future prospects of the biological control of postharvest fruit diseases.

Synthetic fungicides are the primary means to control postharvest diseases of fruits. Biological control has emerged as one of the most promising alternatives to chemicals. During the last 20 years, several biological control agents have been widely investigated for use on different pathogens and fruit crops. Many biological control mechanisms have been suggested for use on fruit including antibiosis, parasitism, induced resistance in the host tissue and competition. With the aim of extending the use of the biofungicides, there have been many studies on the application of various combinations of control agents, and on the application integrated with chemical and physical means of protection. The formulation and application methods are key issues for the efficacy and successful outcome of the commercial product. Genetic engineering may provide a useful tool in the enhancement of the biological control efficacy. Since biofungicides are usually not as effective as pesticides, this approach should be viewed as an important component of an integrated disease management scheme given that a significant and permanent reduction of pesticide use is our goal.

Fusarium oxysporum and its bacterial consortium promote lettuce growth and expansin A5 gene expression through microbial volatile organic compound (MVOC) emission

Fusarium oxysporum MSA 35 [wild-type (WT) strain] is a nonpathogenic Fusarium strain, which exhibits antagonistic activity to plant pathogenic F. oxysporum isolates. The fungus lives in association with a consortium of ectosymbiotic bacteria. The WT strain, when cured of the bacterial symbionts [the cured (CU) form], is pathogenic, causing wilt symptoms similar to those of pathogenic F. oxysporum f. sp. lactucae. Both WT and CU MSA 35 strains produce microbial volatile organic compounds (MVOCs), but with a different spectrum. In vitro dual culture assays were used to assess the effects of the MVOCs produced by WT and CU strains of F. oxysporum MSA 35 on the growth and expansin gene expression of lettuce seedlings. An increase in the root length (95.6%), shoot length (75.0%) and fresh weight (85.8%) was observed only after WT strain MVOCs exposure. Leaf chlorophyll content was significantly enhanced (68%) in WT strain MVOC-treated seedlings as compared with CU strain volatiles and nontreated controls. β-Caryophyllene was found to be one of the volatiles released by WT MSA 35 responsible for the plant growth promotion effect. Semi-quantitative and quantitative reverse transcription-PCR assays indicated a significant difference in the expansin gene expression level between leaf (6.7-fold) and roots (4.4-fold) exposed to WT strain volatiles when compared with the CU strain volatiles and those that were nonexposed.

Alternatives to Methyl bromide in Strawberry Production in the United States of America and the Mediterranean Region

Methyl bromide (MB) is a broad-spectrum soil fumigant, which has been critical in strawberry production for forty years. Strawberry and other high-value cash crops benefit from pre-plant soil fumigation with MB and chloropicrin (Pic). Mixtures of these two fumigants work synergistically in controlling a wide range of plant pathogens and pests, including fungi, nematodes, insects, mites, rodents, weeds, and some bacteria. Methyl bromide was listed in 1993 by the Parties of the Montreal Protocol as an ozone-depleting compound. According to the Montreal Protocol, the import and manufacture of MB in the United States of America (USA) and other developed countries will be banned by 2005, after stepwise reductions in 1999, 2001, and 2003. Currently, there is no single registered alternative fumigant for all of the MB uses and there is a need for environmentally sound and economically feasible alternatives. The fumigants 1,3-dichloropropene (1,3-D) and Pic in combination with methyl isothiocyanate (MITC) generators have shown to be the most promising alternatives to methyl bromide for strawberry production. Studies with the experimental fumigants methyl iodide and propargyl bromide suggested that these compounds have higher reactivity than MB as stand-alone fumigants. This review evaluates the commercially available and experimental alternatives to MB soil fumigation for strawberry production based on relevant scientific publications, proceedings, and personal communications.

Diseases of basil and their management.

weet basil (Ocimum basilicum L.) is an economically important herb crop in several Mediterranean countries. Approximately 80 ha are grown annually in Italy, 30 ha in France (C. Poncet, I.N.R.A., Antibes, France, personal communication), and 20 ha in Israel (T. Katan, ARO Volcani Center, Bet Dagan, Israel, personal communication). Basil is also an important fresh and processed (frozen and pesto sauce) crop in U.S. markets (7,38; A. P. Keinath, Clemson University, Charleston, SC, personal communication). It is used fresh, dried, and processed for flavoring and fragrances and in traditional medicines (5,55). The crop value in Italy can reach

Bacterial ectosymbionts and virulence silencing in a Fusarium oxysporum strain

500,000 per ha per year. Recently, increased interest in so-called “Mediterranean cuisine” has led to a significant increase in basil consumption worldwide. Basil has become associated with the consumption of pasta and with healthy eating habits. It ranks first among the herbs most commonly used by California restaurants (4). In its processed form (pesto), it is known worldwide, thus strongly contributing to the image of Italian cuisine. In Italy, most production takes place in the Riviera Ligure (59) (Fig. 1), where 53 specialized farms with average areas of 1 ha, and about 100 nonspecialized farms with average areas of 0.15 to 0.20 ha, grow basil. All the Ligurian farms are typically family-managed properties. Basil’s high value makes it suitable for farmers with small operations in other countries such as Israel (T. Katan, personal communication) and the United States (57). O. basilicum apparently is native to India (26) and naturalized in Africa and the Pacific Islands (53). However, recent findings show that this species was known in Egypt at the time of the pharaohs (39). Basil has been cultivated in Italy for more than 100 years. It is a member of Labiatae (Ocimoideae) and is related to other herbs, such as mint (Mentha spp.), oregano (Origanum vulgare), marjoram (Origanum majorana), sage (Salvia officinalis), lemon balm (Melissa officinalis), rosemary (Rosmarinus officinalis), and thyme (Thymus serpyllum). In Italy, Genovese Gigante is the basil cultivar most appreciated for fresh consumption. It is the only cultivar used for industrial production of pesto and covers 90% of the total growing area (49). Basil is grown year-round in Italy in the Riviera Ligure, but it is a fall, winter, or spring crop in other locations and latitudes. In Italy, basil typically is grown as a directseeded crop by using 10 to 12 g (1 g = 600 to 800 seeds) of seeds per m2, with planting and management strategies dictated by the market destination (fresh consumption or pesto sauce). The number of plants per m2 can reach 5,000 to 6,000. In Italy, basil is often overseeded after the first two to three harvests of the previous crop and is repeat-harvested after regrowth. In Israel and the United States, basil is transplanted. Most fresh-market basil is hand-harvested and sold as bunches of 10 to 15 plants. Basil for processing often is harvested mechanically. As with most agricultural commodities, diseases impose significant production constraints, affecting both yield and overall quality of basil. Many diseases afflict basil. This article focuses on the most important diseases of basil in Italy, with reference to work carried out by several authors in other regions where basil is grown.

Identification of Race 1 of Fusarium oxysporum f. sp. lactucae on Lettuce by Inter-Retrotransposon Sequence-Characterized Amplified Region Technique

In the present article we have ascertained the presence of a consortium of ectosymbiotic bacteria belonging to Serratia, Achromobacter, Bacillus and Stenotrophomonas genera associated to the mycelium of the antagonistic Fusarium oxysporum MSA 35 [wild-type (WT) strain]. Morphological characterization carried out on the WT strain, on the F. oxysporum MSA 35 without ectosymbionts [cured (CU) strain] and on the pathogenic F. oxysporum f.sp. lactucae (Fuslat 10) showed that the ectosymbionts, present only in the WT strain, caused a depleted production of micro conidia and aerial hyphae, and a change in shape and dimension of the latter. Virulence tests showed that the cured Fusarium was a pathogenic strain and, as shown by polymerase chain reaction and microscope analysis, pathogenicity was correlated with the capability of the cured hyphae of penetrating lettuce roots. Accordingly, the hyphae of the WT strain were impaired in entering the plant roots. Typing experiments provided evidence that both CU and WT strains belong to F. oxysporum f.sp. lactucae. This implies that the antagonistic effect of WT Fusarium is not a fungal trait, but it is due to the interaction with the ectosymbiotic bacteria. Expression analysis showed that fmk1, chsV and pl1 genes involved in F. oxysporum pathogenicity are not expressed in the WT strain whereas they are expressed in the cured fungus. These results, together with the hyphal characteristics, suggest that the inability of WT strain to penetrate the plant roots could be due to alterations in the expression profile of cell wall-degrading enzymes. In conclusion, we demonstrated a modulation of F. oxysporum gene expression in response to the interaction with the ectosymbiotic bacteria. Preliminary researches indicated that the presence of bacteria attached to the hyphae of antagonistic F. oxysporum is not an isolated phenomenon. Further investigations are necessary to better understand the rule and the diffusion of ectosymbiotic bacteria among antagonistic Fusarium.

First Report of Fusarium oxysporum on Lettuce in Europe

ABSTRACT Fusarium wilt of lettuce, caused worldwide by Fusarium oxysporum f. sp. lactucae, is an emerging seed-transmitted disease on Lactuca sativa. In order to develop a molecular diagnostic tool for identifying race 1 (VCG0300) of the pathogen on vegetable samples, an effective technique is presented. Inter-retrotransposon amplified polymorphism polymerase chain reaction (PCR), a technique based on the amplification of genomic regions between long terminal repeats, was applied. It was shown to be useful for grouping F. oxysporum f. sp. lactucae race 1 isolates. Inter-retrotransposon sequence-characterized amplified regions (IR-SCAR) was used to develop a specific set of PCR primers to be utilized for differentiating F. oxysporum f. sp. lactucae isolates from other F. oxysporum isolates. The specific primers were able to uniquely amplify fungal genomic DNA from race 1 isolates obtained in Italy, Portugal, the United States, Japan, and Taiwan. The primers also were specific to pathogen DNA obtained from artificially infected lettuce seed and naturally and artificially infected plants.

Development of a microcantilever-based immunosensing method for mycotoxin detection

In spring 2001, plants of the lettuce cv. Salad Bowl showing symptoms of a wilt disease were observed in several commercial plastic greenhouses near Bergamo, in northern Italy. Wilted plants were first observed during the spring and summer of 2001 when temperatures were between 26 and 35°C. Symptoms were observed in the same area and the same farms in March 2002, in concomitance with a period of high temperatures. Although the distribution of the disease was generally uniform, symptoms were more severe in the central part of the greenhouses where temperatures were warmest. Symptoms were first observed at thinning, when seedlings (30 days old) appeared wilted. Vascular tissues of affected seedlings appeared red or brown. Affected plants were stunted and developed yellow leaves and brown or black streaks in the vascular system. The vascular streaks in the yellow leaves extended from the crown and were continuous with a red-brown discoloration in the vascular system of the crown and upper taproot. Symptoms were typically not visible on the outside of the crowns or roots. Fusarium oxysporum was consistently and readily isolated from symptomatic vascular tissues on a Fusarium-selective medium (2). Seeds of cv. Salad Bowl were planted in steam-sterilized soil artificially infested with 1 × 104 CFU/g soil of each of two isolates of F. oxysporum obtained from infected plants. Plants grown in noninfested soil served as noninoculated control treatments. Plants (25 per treatment) were grown at 25 to 28°C in growth chambers. Wilt symptoms and vascular discoloration of the roots, crown, and veins developed 20 days after seeding for all plants grown in the infested soil, and F. oxysporum was consistently reisolated from infected plants. The pathogenicity test was conducted twice. To our knowledge, this is the first report of F. oxysporum on lettuce in Europe. A wilt of lettuce attributed to F. oxysporum f. sp. lactucae was reported in Japan in 1967 (3) and in the United States in 1993 (1), in the latter case the incitant organism was described as F. oxysporum f. sp. lactucum. References: (1) J. C. Hubbard and J. S. Gerik. Plant Dis. 77:750, 1993. (2) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (3) T. Matuo and S. Motohashi. Trans. Mycol. Soc. Jpn. 8:13, 1967.

First report of brown rot of stone fruit caused by Monilinia fructicola in Italy.

Mycotoxins, such as aflatoxins and ochratoxin A, are presently considered as the most important chronic dietary risk factor, more than food additives or pesticide residues. Therefore, the serious health and economic consequences of mycotoxin contamination have created the need for rapid, sensitive, and reliable techniques to detect such dangerous molecules within foodstuffs. We here report on the development of an innovative immunosensing method for mycotoxin detection, based on antibody-immobilized microcantilever resonators, a promising label free biosensing technique. A considerable part of the work is devoted to show the effect on microcantilever resonance frequency of the composition of the incubation buffer, as well as of the washing and drying procedure. We show the feasibility of using microcantilever resonator arrays to effectively identify total aflatoxins and ochratoxin A, at low concentrations (3 ng/mL and less than 6 ng/mL, respectively), with relatively low uncertainty (about 10%) and good reproducibility for the same target concentration. Furthermore, the developed immunosensing method shows a limited cross-reactivity to different mycotoxins, paving the way to a highly specific technique, able to identify different mycotoxins in the sample. To our knowledge, this work represents the first example in literature of successfully immunodetection of low concentrations of multiple mycotoxins by microcantilever resonator arrays.

A new method for detection of five alternaria toxins in food matrices based on LC–APCI-MS

Monilinia fructicola, causal agent of brown rot, is one of the most important fungal pathogens of stone fruit. M. fructicola is a quarantined pathogen in Europe. During the summer of 2008 in 15 orchards located in Piedmont (northern Italy), 12,500 stone fruits (cherries, apricots, peaches, nectarines, and plums) were stored in cold chambers at 4 and 6°C and monitored for 8 weeks for the presence of Monilinia spp. M. fructicola was detected on 0.5% of nectarines (cvs. Sweet Red and Orion) that originated from two orchards in Lagnasco. Symptoms appeared on the fruit during storage, starting 3 weeks after harvest. Fruit rot lesions were brown, sunken, and covered with grayish tufts. The majority of infected fruit became dry and mummified. Brown rot symptoms were similar to those caused by endemic M. fructigena and M. laxa. Symptoms began with a small, circular, brown spot, and the rot spread rapidly. At the same time, numerous, small, grayish stromata developed. Finally, the whole surface of the fruit was covered by conidial tufts. Tissues were excised from diseased stone fruits and cultured on potato dextrose agar (PDA) amended with 25 μg of streptomycin per liter. The isolates produced abundant mycelium on PDA at 20 ± 2°C. Colonies were initially gray, but after sporulation, they became hazel, showing concentric rings (sporulation is sparse in M. laxa or M. fructigena). Conidia were one-celled, ellipsoid, hyaline, 15.2 × 10.1 μm, and produced in branched monilioid chains (2). Preliminary morphological identification of fungi resembling M. fructicola was confirmed by PCR using genomic DNA extracted from the mycelia of pure cultures. The DNA was amplified with a common reverse primer and three species-specific forward primers (3) obtained from a sequence characterized amplified region and a product of 535 bp, diagnostic for the species M. fructicola, was obtained. BLAST analysis of the amplified sequence (GenBank Accession No. FI569728) showed 96% similarity to the sequence of a M. fructicola isolated from Canada (GenBank Accession No. AF506700), 15% similarity to M. laxa ATCC11790 (GenBank Accession No. AF506702), and 35% similarity to a M. fructigena sequence isolated in Italy (GenBank Accession No. AF506701). Moreover, two sequences obtained through the amplification of ribosomal region ITS1-5.8S-ITS2, showing 100% similarity to the same ribosomal sequence of M. fructicola, were deposited in GenBank (Accession Nos. FJ411109 and FJ411110). The pathogen was detected on some mummified fruit from the same orchards in November of 2008. Pathogenicity was tested by spraying 103 conidia/ml on 10 surface-sterilized artificially wounded nectarines per strain of M. fructicola. After 5 days of incubation at 20 ± 2°C, typical, brown, rot symptoms developed on inoculated fruit. M. fructicola was reisolated from the inoculated fruit on PDA. Symptoms did not appear on control fruit. To our knowledge, this is the first report of M. fructicola in Italy. Its occurrence in Europe has been reported sporadically in Austria and France, and in 2006, it was detected in Hungary and Switzerland on peaches and nectarines imported from Italy and Spain (1,4). References: (1) E. Bosshard et al. Plant Dis. 90:1554, 2006. (2) R. J. W. Byrde and H. J. Willetts. The Brown Rot Fungi of Fruit: Their Biology and Control. Pergamon Press, Oxford, 1977. (3) M. J. Coté et al. Plant Dis. 88:1219, 2004. (4) M. Petròczy and L. Palkovics. Plant Dis. 90:375, 2006.