LIBRO-CONGRESO-CITRUS

SOD activity in the Δsod1 mutant was approximately 50% of the wild type. Furthermore, since cell invasion by
P. digitatum is pH-dependent, we also investigated the pH-dependent transcription factor PacC. The ΔPacC
mutant almost completely lost its pathogenicity towards mature fruit, suggesting that PacC also contributes
to the P. digitatum pathogenicity.
S09P18
Identification and expression analysis of Penicillium digitatum genes involved in fungal virulence
during citrus fruit infection
de Ramón-Carbonell M. 1 , Ballester A.R. 2 , González-Candelas L. 2 , and Sánchez-Torres P. 1
1 Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro de Protección Vegetal y Biotecnología PVyB, Spain; and 2 Instituto de
Agroquímica y Tecnología de Alimentos (IATA), Fisiología y Biotecnologíam Postcosecha, Spain. palomas@ivia.es
Penicillium digitatum, the causal agent of Citrus Green Mold Rot, is the major pathogen of citrus fruit during
postharvest. The control of this pathogen relies on the use of fungicides. However, due to the appearance of
resistant strains and the harmful effects of synthetic fungicides on human health and the environment, there
is a need to adopt new and safer control methods. Our knowledge of the mechanisms underlying pathogen’s
virulence in postharvest pathogenic fungi, especially in Penicillium-fruit interactions, is still very poor. The aim
of this study was to identify genes expressed in a virulent P. digitatum strain (Pd1) during the early stages of
citrus fruit infection through suppression subtractive hybridization (SSH), using a low virulence P. digitatum
isolate (CECT2954) as a driver. The subtracted cDNA library was sequenced using 454-FLX Titanium technology
to perform a global transcriptomic analysis of putative fungal virulence genes. Gene expression analysis of
selected P. digitatum genes putatively involved in fungal virulence was carried out by qRT-PCR. The expression of
these genes was analyzed in both the high and the low virulent P. digitatum strains in three different situations:
i) in orange disks after 24 h of P. digitatum interaction, ii) during growth of P. digitatum strains in PDB medium
from 24 to 72 h and iii) in orange fruits during P. digitatum strains infection from 24 to 72 h. qRT-PCR profiling of
selected fungal genes revealed different expression patterns during infection of orange fruits.
S09P19
Metabolism of flavonoids and mycotoxins in citrus fruits infected by Alternaria alternata
Del Río J.A. 1 , Díaz L. 1 , Ortuño A. 1 , García-Lidón A. 2 , and Porras I. 2
1 Faculty of Biology. University of Murcia. (UMU), Plant Biology, Murcia (Spain); and 2 Murcian Institute of Agriculture and Food
Research and Development (IMIDA), Citriculture, Murcia (Spain). ignacio.porras@carm.es
Fungi of the genus Alternaria are responsible for substantial pre-harvest losses in citrus. The susceptibility to
A. alternata depends on the citrus species. In this communication, the metabolism of Alternaria mycotoxins
and citrus flavonoids are analyzed in leaves and fruits of different citrus species and cultivars. HPLC-MS study
revealed the presence of two A. alternata mycotoxins involved in the evolution of brown spot in leaves
and fruits. These were identified as 3,7,9- trihydroxy-1-methyl-6H-dibenzo [b,d] piran-6-ona, known as
alternariol (AOH), and 3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d] piran-6-ona, known as alternariol
monomethyl ether (AME). Biological assays involving the inoculation of leaves and fruits with the above
mentioned toxins showed that both molecules caused necrosis in the inoculated citrus tissues, suggesting
that the mycotoxins may be involved in the evolution of necrotic spot caused by A. alternata. The increased
expression of the above mycotoxins was associated with the end of mycelial growth, high sporulation and
an increase in hyphal melanisation. Citrus flavanones and flavones such as hesperidin, naringin, diosmin and
sinensetin increased the biosynthesis of these micotoxins. On the other hand, a degradation of flavonoids
was observed when leaves and fruits were infected with A. alternata. A decrease in the concentration of the
flavanones hesperidin, naringin and their respective aglicons was observed in leaves and fruits of susceptible
citrus. An increase of extracellular proteins with oxidant activity was detected in leaves and fruits inoculated
with Alternaria. The study of substrate specificity, molecular weight and different inhibitors confirmed that
Alternaria expressed an extracellular laccase, and that the citrus flavonoids are substrates of this enzyme. The
involvement of mycotoxins, laccase enzyme and degradation of flavonoids in the pathogenesis of A. alternata
in citrus is discussed.
XII INTERNATIONAL CITRUS CONGRESS 2012 - 145
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