Nicola Luchi

Real-time PCR detection of Biscogniauxia mediterranea in symptomless oak tissue

Aims: Real‐time PCR, based on TaqMan® chemistry, was used to detect Biscogniauxia mediterranea, a fungal pathogen that after a long endophytic phase may cause charcoal disease in oak trees.

New proteins orthologous to cerato-platanin in various Ceratocystis species and the purification and characterization of cerato-populin from Ceratocystis populicola

Natural variants of cerato-platanin (CP), a pathogen associated molecular pattern (PAMP) protein produced by Ceratocystis platani (the causal agent of the plane canker stain), have been found to be produced by other four species of the genus Ceratocystis, including five clones of Ceratocystis fimbriata isolated from different hosts. All these fungal strains were known to be pathogenic to plants with considerable importance in agriculture, forestry, and as ornamental plants. The putative premature proteins were deduced on the basis of the nucleotide sequence of genes orthologous to the cp gene of C. platani; the deduced premature proteins of Ceratocystis populicola and Ceratocystis variospora reduced the total identity of all the others from 87.3% to 60.3%. Cerato-populin (Pop1), the CP-orthologous protein produced by C. populicola, was purified and characterized. Pop1 was a well-structured α/β protein with a different percentage of the α-helix than CP, and it self-assembled in vitro in ordered aggregates. Moreover, Pop1 behaved as PAMP, since it stimulated poplar leaf tissues to activate defence responses able to reduce consistently the C. populicola growth.

Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

ABSTRACT Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10−2 to 1.4 × 10−2 pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management.

Early detection of Biscogniauxia nummularia in symptomless European beech (Fagus sylvatica L.) by TaqManTM quantitative real‐time PCR

Aims:  To develop a quantitative real‐time PCR (Rt PCR) assay for the early detection of Biscogniauxia nummularia, a xylariaceous fungus that causes strip‐canker and wood decay on European beech (Fagus sylvatica L.).

Ecology of invasive forest pathogens

Invasive forest pathogens are a major threat to forests worldwide, causing increasing damage. The knowledge of both the specific traits underlying the capacity of a pathogen to become invasive, and the attributes predisposing an environment to invasion are to be thoroughly understood in order to deal with forest invasions. This paper summarizes the historical knowledge on this subject. Many aspects of the ecological processes underlying alien forest pathogens invasions are still unknown, which raises several scientific issues that need further study. The introduction of invasive forest pathogens to areas where naïve hosts are found, is mainly due to global plant trade. Rapid transportation and reduced delivery times increase the chances of survival of pathogen propagules and of their successful establishment in new environments. In forest pathogens, the reproduction mode seems not to be a crucial determinant of invasiveness, as highly destructive pathogens have a variety of reproductive strategies. The most important drivers of forest pathogen invasions appear to be (a) great adaptability to new environmental conditions; (b) efficient dispersal over long and short distances, possibly assisted by the capacity to form novel associations with endemic and/or alien insect vectors; (c) the ability to exchange genetic material or hybridize with resident or alien species. Moreover, these features interact with some key traits of the invaded environment, e.g. environmental variability and biodiversity richness. Host resistance and natural enemies may occur as a result of rapid selection/adaptation after the epidemic phase of invasion.

Early physiological responses of Pinus pinea L. seedlings infected by Heterobasidion sp.pl. in an ozone-enriched atmospheric environment

The presence of the American root-rot disease fungus Heterobasidion irregulare Garbel. & Otrosina was detected in Italian coastal pine forests (Pinus pinea L.) in addition to the common native species Heterobasidion annosum (Fries) Brefeld. High levels of tropospheric ozone (O3) as an atmospheric pollutant are usually experienced in Mediterranean pine forests. To explore the effect of interaction between the two Heterobasidion species and ozone pollution on P. pinea, an open-top chamber (OTC) experiment was carried out. Five-year-old P. pinea seedlings were inoculated with the fungal species considered (H. irregulare, H. annosum and mock-inoculation as control), and then exposed in charcoal-filtered open-top chambers (CF-OTC) and non-filtered ozone-enriched chambers (NF+) from July to the first week of August 2010 at the experimental facilities of Curno (North Italy). Fungal inoculation effects in an ozone-enriched environment were assessed as: (i) the length of the inoculation lesion; (ii) chlorophyll a fluorescence (ChlF) responses; and (iii) analysis of resin terpenes. Results showed no differences on lesion length between fungal and ozone treatments, whereas the short-term effects of the two stress factors on ChlF indicate an increased photosynthetic efficiency, thus suggesting the triggering of compensation/repair processes. The total amount of resin terpenes is enhanced by fungal infection of both species, but depressed by ozone to the levels observed in mock-inoculated plants. Variations in terpene profiles were also induced by stem base inoculations and ozone treatment. Ozone might negatively affect terpene defences making plants more susceptible to pathogens and insects.

Production of Diplodia scrobiculata and diplodia pinea pycnidia on ground Austrian pine needle agar medium

The in vitro production of fruiting structures represents an important tool for the morphological identification of fungal genera or species. It is also important for the controlled production of spores to be used in experiments. However, some fungal species do not readily sporulate in pure culture. In the present study we induced the production of pycnidia of Diplodia scrobiculata and D. pinea, two species recalcitrant to sporulation in pure culture, by growing them on two media containing ground Austrian pine needle. The two fungal species grew equally rapidly on both media and pycnidial primordia were produced on the medium surface after only 4 days at room temperature. Conidia matured in less than two weeks and their germination rate at 25°C was about 96%, indicating high viability.

Laser microdissection on Norway spruce bark tissue: A suitable protocol for subsequent real-time reverse transcription–polymerase chain reaction (RT-PCR) analysis

Abstract The development of new tools able to select specific plant tissue is crucial for gene expression studies. During the last years, the use of laser microdissection, mainly tested on herbaceous plant tissue, has been found to be a useful technique for these purposes. This method is poorly tested on woody species, and so far no studies of gene expression have been applied on forest trees. For this reason the present work proposes the optimization of a functional protocol using laser microdissection pressure catapulting (LMPC) and real-time reverse transcription–polymerase chain reaction (RT-PCR) in bark stem tissue of Norway spruce (Picea abies). Bark tissue fragments were collected from Norway spruce trees and sliced with a cryostat. RNA was extracted from both whole cross-sections and microdissected bark cells. The feasibility of the method was confirmed by the amplification of the α-tubulin, an endogenous gene of P. abies, with efficiency comparable to that obtained from non-microdissected tissue. The proposed protocol, here adapted for bark tissue of woody species, represents a useful tool in a wide range of hosts that, unlike herbaceous plants, have scarcely been considered up to now.

Occurrence of Pythium and Phytopythium species isolated from citrus trees infected with gummosis disease in tunisia

Abstract A survey was conducted, during 2012/2013, to identify the causal agents of citrus gummosis in Tunisia. The obtained isolates were identified by molecular and pathological criteria. Three species of Pythium (P. aphanidermatum, P. ultimum and P. dissotocum) and two species of Phytopythium (P. vexans and P. mercuriale) were recovered. Pathogenic tests, using twigs inoculation method for five citrus varieties under laboratory conditions, showed necrotic lesions similar to those observed in natural infected citrus trees by gummosis. In greenhouse experiments, the pathogenicity of the species to citrus was demonstrated by soil and trunk inoculations. Results showed necrotic lesions in the trunk of the variety “Clementine”. P. ultimum is the most virulent, while P. mercuriale was the least virulent in trunk inoculation. Symptom of necrotic roots has been also observed. This is the first report of Pythium and Phytopythium species within the citrus area in Tunisia.

Phytophthora nicotianae and P. cryptogea causing gummosis of citrus crops in Tunisia

Surveys were conducted in 2013/2014 across seven Tunisian citrus production areas for the identification of pathogens associated with citrus gummosis. Samples were analyzed using PARP-BH selective media. Collected isolates were identified based on morphology and DNA sequences of the internal transcribed spacer (ITS) region analysis. Ten isolates of P. nicotianae and five isolates of P. cryptogea were obtained. In vitro inoculation of five citrus varieties (Clementine Hernandina, Mandarins Tangerine, Blood Orange Maltese, Valencia Late and Thomson Navel) showed that P. nicotianae were pathogenic to cuttings twigs and P. cryptogea were pathogenic to leaves. In the case of P. nicotianae, the most susceptible variety was C. hernandina, while the most tolerant variety was M. tangerine. For P. cryptogea the most susceptible variety was C. Hernandina, while the most tolerant variety was V. late. In greenhouse assays, inoculation of trunks of C. Hernandina with P. nicotianae and P. cryptogea inocula induced the appearance of necrotic area and gum-exudation when inoculated with P. nicotianae. However, inoculation of rootstocks Sour orange with the same isolates induced root necrosis and leaf wilt. This is the first report of P. cryptogea causing gummosis in citrus orchards in Tunisia.