Camilla Martini

Biological control of postharvest diseases by microbial antagonists: how many mechanisms of action?

The postharvest phase has been considered an environment for successful application of biological control agents (BCAs). However, the interactions between fungal pathogen, host (fruit), and antagonist are influenced by several parameters such as temperature, oxidative stresses, oxygen composition and water activity that could determine the success of biocontrol. Knowledge of the modes of action of BCAs is essential in order to enhance their viability and increase their potential in disease control. The antagonists display a wide range of modes of action: antibiosis, competition for nutrients and space, parasitism and induction of resistance are considered the main ones. Their efficacy, however, is related to the host and the pathogen; sometimes, these mechanisms could act simultaneously, and it is therefore difficult to establish which is related to a specific antifungal action. The current review presents a brief summary of the research that has led to a better understanding of the mode of action of BCAs with particular emphasis on the most recent literature.

Control of postharvest fungal pathogens by antifungal compounds from Penicillium expansum.

The fungicidal effects of secondary metabolites produced by a strain of Penicillium expansum (R82) in culture filtrate and in a double petri dish assay were tested against one isolate each of Botrytis cinerea, Colletotrichum acutatum, and Monilinia laxa and six isolates of P. expansum, revealing inhibitory activity against every pathogen tested. The characterization of volatile organic compounds released by the R82 strain was performed by solid-phase microextraction-gas chromatographic techniques, and several compounds were detected, one of them identified as phenethyl alcohol (PEA). Synthetic PEA, tested in vitro on fungal pathogens, showed strong inhibition at a concentration of 1,230 μg/ml of airspace, and mycelium appeared more sensitive than conidia; nevertheless, at the concentration naturally emitted by the fungus (0.726 ± 0.16 m g/ml), commercial PEA did not show any antifungal activity. Therefore, a combined effect between different volatile organic compounds produced collectively by R82 can be hypothesized. This aspect suggests further investigation into the possibility of exploiting R82 as a nonchemical alternative in the control of some plant pathogenic fungi.

Activities of Aureobasidium pullulans cell filtrates against Monilinia laxa of peaches

The Aureobasidium pullulans L1 and L8 strains are known as efficient biocontrol agents against several postharvest fungal pathogens. In order to better understand the mechanism of action underneath the antifungal activity of L1 and L8 strains, yeast cell filtrates grown at different times were evaluated in vivo against Monilinia laxa on peach. Lesion diameters on peach fruit were reduced by L1 and L8 culture filtrates of 42.5% and 67% respectively. The ability of these filtrates to inhibit M. laxa conidia germination and germ tube elongation was studied by in vitro assays. The results showed a 70% reduction of conidia germination for both strains while for germ tube elongation, it was 52% and 41% for L1 and L8 culture filtrates respectively. Finally, the activity of cell wall hydrolytic enzymes such as chitinase and glucanase in cell filtrates was analysed and the expression of genes encoding these activities was quantified during yeast growth. From 24h onward, both culture filtrates contained β,1-3,glucanase and. chitinase activities, the most pronounced of which was N-β-acetylglucosaminidase. Gene expression level encoding for these enzymes in L1 and L8 varied according to the strain. These results indicate that L1 and L8 strains culture filtrates retain the yeast antagonistic activity and suggest that the production of hydrolytic enzymes plays an important role in this activity.

First report of Colletotrichum acutatum causing bitter rot on apple in Italy.

Italy could be considered the main apple-producing country in the European Union. Italian apple (Malus domestica L. Borkh.) production is estimated at approximately 2.1 million tons and encompasses a wide range of cultivars, harvested from August to November. Colletotrichum acutatum, which causes severe losses to strawberry production, was a regulated organism for all European countries until 2008, when it was removed from the EPPO quarantine pathogen list because of its wide distribution in strawberry-production areas. During the growing season of 2010, fungi were isolated from apple fruits exhibiting bitter rot symptoms after 4 months of storage in several packinghouses in the Emilia Romagna Region. The apples belonged to the Golden Delicious, Granny Smith, Pink Lady and Crisp Pink cultivars. Lesions on the fruit surface were circular and 1 to 3 cm in diameter. When lesions enlarged, they became sunken with relatively firm rotten tissues. The fungal fruiting structures, acervuli, were distributed sparsely or densely on old lesions, and under humid conditions, they discharged an orange conidial mass. Conidia observed with a light microscope appeared hyaline and fusiform, 8 to 16 × 2.5 to 4 μm, with two pointed ends or one rounded end. The fungal isolates were grown on potato dextrose agar (PDA) plates incubated at 25°C. After 7 days, colonies were white, becoming gray to pale orange, and when viewed from the reverse side, the color ranged from pink to reddish orange. Both cultural and morphological characteristics of the pathogen were similar to those described for C. acutatum J.H. Simmonds (3), which is responsible for bitter rot of apple (2). Kochs postulates were performed with one representative isolate from each host by artificial inoculation of 30 healthy apples from the cultivars listed above. Fruit surfaces were disinfected with 70% ethanol, wounded with a sterile needle, and then inoculated with 20 μl of a spore suspension (105 conidia ml-1) prepared from a 15-day-old culture on PDA. Inoculated fruits were sealed in a plastic bag and incubated at 25°C for 10 days. In 92% of fruits, symptoms appeared 10 days later, forming lesions with cream-to-salmon pink fruiting structures. The fungus was reisolated onto PDA from the lesions on the inoculated apples. After 7 days of incubation, the colonies and the morphology of conidia were the same as those of the original isolates. The tests were performed on all four cultivars with similar results. The PCR analysis, carried out using universal primers ITS1 and ITS4 (4) directly from single-spore-derived mycelium (1), resulted in an amplification product with 100% sequence homology with C. acutatum isolate AB626881 from GenBank database. Considering the results obtained, to our knowledge, this is the first report of C. acutatum in Italy causing bitter rot on apple. The disease is common in practically all countries where apples are commercially grown and since the losses could be severe under prolonged warm and wet weather conditions, C. acutatum could represent a serious issue for the Italian apple industry. References: (1) M. Iotti and A. Zambonelli. Mycol. Res. 110:60, 2006. (2) A. L. Jones et al. Plant Dis. 80:1294, 1996. (3) B. C. Sutton. Page 1 in: Colletotrichum: Biology, Pathology and Control. Brit. Soc. Plant Pathol. Oxon. UK 1992. (4) T. J. White et al. Page 315 in: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Monilinia fructicola , Monilinia laxa (Monilinia Rot, Brown Rot)

Abstract Monilinia spp. causing brown rot are fungal pathogens producing severe losses in worldwide stone fruit production, with high economic relevance (M€ 1.7/year). There are three main fungal species: Monilinia laxa , M. fructigena and M. fructicola . The pathogen infects aerial parts of host plants with a variety of symptoms, including blighting of blossoms, cankers on woody tissues and rotting of fruits, although the prevalent fruit losses are in the postharvest phase. Fungicide sprays in pre- and postharvest periods are usually used to control Monilinia spp. but, in Europe, no fungicide fruit treatments are allowed after harvest. Many studies conducted in recent years have defined three main research fields within the alternative methods for postharvest brown rot control: (1) biological control sensu strictum with microbial antagonists; (2) use of natural bioactive compounds; and (3) use of physicochemical methods. Although the reviewed results have reported a significant reduction of pesticide use, a multidisciplinary approach is recommended in the future, where biological products will be combined with low-risk chemical fungicides, natural antimicrobial substances and other physical means for an integrated strategy to control brown rot.

CHARACTERIZATION OF THIOPHANATE METHYL RESISTANCE IN ITALIAN MONILINIA FRUCTICOLA ISOLATES

Monilinia fructicola causes considerable damage to cultivated stone fruits in the temperate regions with an important economic impact. Monitoring the strains resistant to fungicides is essential to reduce economic losses associated with the peach and nectarine market. Although several works have focused on benzimidazole fungicide resistance worldwide in Monilinia spp., limited data report the benzimidazole resistance in European M. fructicola isolates. In order to assay the development of resistance to thiophanate methyl, the Alamar Blue test, a quick and reliable assay, was used and the results compared with those obtained with conventional amended medium. Our results show for the first time the presence in Italian M. fructicola isolates of a phenotype resistant to thiophanate methyl. In particular, 46 out of 63 isolates were found resistant, with EC50 values ranging from 0.99 μg ml-1 to 57.59 μg ml-1, values equal or higher than the inhibitory dose (1 μg ml-1). Point mutations in the β-tubulin gene were analyzed in 18 representative M. fructicola isolates, 15 with different levels of resistance (low and high resistance) and three sensitive. All resistant isolates tested showed a point mutation at codon 198 with respect to sensitive isolates isolates, i.e. GCA instead of GAA. In addition, all Italian isolates revealed a point mutation at codon 83 in the β-tubulin gene where the arginine was converted to glutamine with a punctual allelic change CAA instead of CGA.