Journal of Plant Pathology (2010), 92 (4, Supplement ... - Sipav.org

S4.86 Journal of Plant Pathology (2010), 92 (4, Supplement), S4.71-S4.105
These isolates were identified by morphological and molecular
methods. Fungal DNA was subjected to RAPD (Random Amplified
Polymorphic DNA) analysis using 20 decamer primers. A total
of 248 markers (from 209 to 1,800 bp in size) were obtained.
The number of RAPD markers generated per primer varied between
15 and 28. Similarity matrix using Dice coefficient for pairwise
comparisons gave a dendrogram that grouped the isolates in
clusters corresponding to the anamorph. A broad inter-specific
variability resulted, each species being well distinguished from
the most closely related ones. RAPD analysis confirmed to be a
reliable technique for investigating species differentiation and genetic
diversity. The technique can be of help in the identification
of some Botryosphaeriaceae species, being cheaper and faster
than the commonly used methods based on sequencing of ITS region,
often integrated with partial sequencing of EF1-α or βtubulin
genes.
DEVELOPMENT OF A MACROARRAY SYSTEM FOR
THE DIAGNOSIS OF ROOT ROT AND TRACHEOMYCO-
SIS OF ORNAMENTAL PLANTS. A. Haegi 1,2 , M. Paoli 3 , D.
Rizzo 3 , L. Riccioni 2 , A. Grassotti 1 . 1 CRA, Unità di Ricerca per il
Vivaismo e la Gestione del Verde Ambientale ed Ornamentale
(CRA-VIV), Via dei Fiori 8, 51012 Pescia (PT), Italy. 2 CRA, Centro
di Ricerca per la Patologia Vegetale (CRA-PAV), Via C.G. Bertero
22, 00156 Roma, Italy. 3 ARSIA, Via dei Fiori 8, 51012 Pescia
(PT), Italy. E-mail: anita.haegi@entecra.it
The industry of ornamental plants is characterized by continuous
introduction of new crops and new production technologies
that creates new opportunities for pathogens to exploit. In this
framework an early and precise identification of pathogens is critical
for determining defence strategy. In this work we addressed
root rot and tracheomycosis diseases of perennial ornamental
plants. Since phytosanitary surveys show an epidemiological complex
situation, a macroarray device was chosen because it can detect
multiple pathogens in a single assay, is sensitive, rapid and
does not require a sophisticated equipment. Surveys were conducted
in some Tuscan farms growing ornamentals to assess the
main phytosanitary problems. Samples were collected and the
fungi isolated from infected tissues were identified by morphology
and molecular tools. On the same samples, a DNA extraction
protocol from infected plant material have been set up, and the
resulting DNA samples were amplified with ITS5/ITS4 primers
for fungi and ITS6/ITS4 primers for oomycetes. The technique
for macroarray procedure was set up using direct labelling (Gene
Images AlkPhos, Amersham). Briefly, oligonucleotide detectors
were immobilized on a nylon membrane and hybridized with the
sample DNA, previously amplified and labelled. For each
pathogen to be tested oligonucleotide detectors were looked for
in the literature and validated or designed ex novo. Oligonucleotide
detectors for Fusarium oxysporum, Phytophthora spp.
and Pythium spp. were found in the literature and are now under
validation. Four oligo detectors for Phytophthora cactorum have
also been designed.
IN VITRO STUDY OF FUM GENES EXPRESSION AND
FUMONISINS PRODUCTION IN FUSARIUM VERTICIL-
LIOIDES UNDER DIFFERENT ECOLOGICAL CONDI-
TIONS. I. Lazzaro 1 , A. Susca 2 , G. Mulè 2 , A. Ritieni 3 , P. Battilani
1 . 1 Istituto di Entomologia e Patologia Vegetale, Università Cattolica
del Sacro Cuore, Via Emilia Parmense 84, 29100 Piacenza,
Italy. 2 Istituto di Scienze delle Produzioni Alimentari del CNR, Via
Amendola 122/O, 70126 Bari, Italy. 3 Istituto di Scienze degli Ali-
menti, Università degli Studi “Federico II”, 80055 Napoli, Italy. Email:
paola.battilani@unicatt.it
F. verticillioides, a fungus associated with maize ears all over
the world, induces Fusarium pink ear rot and produces fumonisins
(FBs), mycotoxins found in maize kernels and their derivatives.
FBs are toxic to humans, FB 1 in particular, which is classified
as possibly carcinogenic. Fumonisins biosynthetic pathway is
regulated by several genes belonging to the FUM cluster whose
behaviour in different ecological conditions is poorly studied.
The aim of this work was to investigate the behaviour of two F.
verticillioides strains grown in vitro both on FB-inducing (Malt
extract agar, MEA) and FB-inhibiting (Czapeck yeast agar, CYA)
media. Fungal growth, FBs production and gene expression were
studied at different temperature (T; 20-30) and water activity (a w;
0.90-0.99) regimes, in liquid cultures incubated for 7-21 days.
The expression of two genes, FUM21 and FUM2, was considered;
the relative quantification of gene expression was performed
by Real time PCR. Results showed that, with a w fixed at
0.99, maximum FUM21 and FUM2 expression was at 30°C after
14 days of incubation, while FBs production increased from 7 to
21 days. At fixed T (25°C), fungal growth at 0.90 a w was not observed
after 21 days of incubation. Gene expression increased
with incubation time both at 0.95 and 0.99 a w ; FB production increased
till 21 and 14 days respectively at 0.95 and 0.99 a w . In all
conditions studied the expression of the two genes considered
followed a very similar trend, but FUM2 was much more expressed
than FUM21 and more influenced by a w conditions.
TRICHODERMA VIRIDE AND PHOMOPSIS sp. ASSOCI-
ATED WITH A DECLINE OF PINUS NIGRA PLANTLETS
IN A REFORESTATION AREA OF CENTRAL ITALY. M.G.
Li Destri Nicosia, S. Mosca, G.E. Agosteo, R. Mercurio, L.
Schena. Dipartimento di Gestione dei Sistemi Agrari e Forestali,
Università degli Studi Mediterranea, Località Feo di Vito, 89122
Reggio Calabria, Italy. E-mail: lschena@unirc.it
In winter 2009, a decline was observed on 4-year-old plantlets
of Pinus nigra in a reforestation area of Abruzzo National Park
(central Italy). More than 70% of the plantlets died during the
first year after transplanting while survivors were stunted and
showed leaf chlorosis as well as root and crown rot. Four fungal
morphotypes, grouped according to the morphology of colonies
and reproductive structures, were isolated from the necrotic subcortical
tissues of the basal stem of symptomatic plantlets. ITS regions
of representative isolates of each morphotype were examined
by BLAST analysis and compared with available sequences
in GenBank. Three isolates were identified as Phomopsis sp., Trichoderma
viride and T. harzianum, respectively, based on a 99-
100% identity with deposited sequences. Conversely, a morphotype
that did not produce conidia was not identified since, surprisingly,
its ITS sequence matched sequences of multiple taxonomic
groups. Pathogenicity tests were conducted on both cuttings
and potted 4-year-old plantlets of P. nigra by inserting a
plug of PDA with actively growing mycelium under the bark. T.
viride and Phomopsis sp. caused necrosis of subcortical tissues
around the inoculation site. Lesions caused by T. viride were significantly
more extended than those caused by Phomopsis sp.
Both fungi were reisolated from symptomatic tissues. No symptoms
were observed on cuttings and plantlets inoculated with
sterile agar, T. harzianum or the unidentified fungus. Both Phomopsis
sp. and T. viride were reported previously as tree
pathogens. However their actual role in the decline of P. nigra
should be further investigated.