Silvana De Leonardis

Defense responses to mycotoxin-producing fungi Fusarium proliferatum, F. subglutinans, and Aspergillus flavus in kernels of susceptible and resistant maize genotypes

Developing kernels of resistant and susceptible maize genotypes were inoculated with Fusarium proliferatum, F. subglutinans, and Aspergillus flavus. Selected defense systems were investigated using real-time reverse transcription-polymerase chain reaction to monitor the expression of pathogenesis-related (PR) genes (PR1, PR5, PRm3, PRm6) and genes protective from oxidative stress (peroxidase, catalase, superoxide dismutase and ascorbate peroxidase) at 72 h postinoculation. The study was also extended to the analysis of the ascorbate-glutathione cycle and catalase, superoxide dismutase, and cytosolic and wall peroxidases enzymes. Furthermore, the hydrogen peroxide and malondialdehyde contents were studied to evaluate the oxidation level. Higher gene expression and enzymatic activities were observed in uninoculated kernels of resistant line, conferring a major readiness to the pathogen attack. Moreover expression values of PR genes remained higher in the resistant line after inoculation, demonstrating a potentiated response to the pathogen invasions. In contrast, reactive oxygen species-scavenging genes were strongly induced in the susceptible line only after pathogen inoculation, although their enzymatic activity was higher in the resistant line. Our data provide an important basis for further investigation of defense gene functions in developing kernels in order to improve resistance to fungal pathogens. Maize genotypes with overexpressed resistance traits could be profitably utilized in breeding programs focused on resistance to pathogens and grain safety.

Constitutive expression of pathogenesis-related proteins and antioxydant enzyme activities triggers maize resistance towards Fusarium verticillioides

Fusarium verticillioides is a fungal pathogen of maize that causes ear rot and contaminates the grains with fumonisin mycotoxins. Breeding for resistance to Fusarium emerged as the most economic and environmentally safe strategy; therefore the discovery of resistant sources and effective molecular markers are a priority. Ears of resistant (CO441 and CO433) and susceptible (CO354 and CO389) maize lines were inoculated with F. verticillioides and the expression of pathogenesis-related (PR) genes (PR1, PR5, PRm3, PRm6) and genes that protect from oxidative stress (peroxidase, catalase, superoxide dismutase and ascorbate peroxidase) were evaluated in the kernels at 72h post inoculation. In addition, the oxidation level and the enzymatic activity of ascorbate-glutathione cycle, catalase, superoxide dismutase and cytosolic and wall peroxidases were investigated. The uninoculated kernels of the resistant lines showed higher gene expression and enzymatic activities, highlighting the key role of constitutive resistance in limiting pathogen attack. In contrast, the susceptible lines activated defensive genes only after pathogen inoculation, resulting in increased levels of H2O2 and lipid peroxidation, as well as lower enzymatic activities. The constitutive defenses observed in this study from seed could be profitably exploited to develop markers to speed up conventional breeding programs in the selection of resistant genotypes.

Increase of fumonisin b2 and ochratoxin a production by black Aspergillus species and oxidative stress in grape berries damaged by powdery mildew.

Powdery mildew (PM), caused by the fungus Erysiphe necator, is one of the most widespread fungal disease of grape and may cause extensive openings on the berry surface during the infection. We evaluated the effect of damage caused by PM in grape berries on the growth of and mycotoxin production by Aspergillus and on the oxidative stress in infected berries. Berries of Vitis vinifera L. cv. Negroamaro with sound skin (SS) and those naturally infected by PM were surface sterilized and inoculated with either fumonisin B2(FB2)-producing strains of Aspergillus niger or ochratoxin A (OTA)-producing strains of Aspergillus carbonarius and incubated at 20 and 30°C. The PM berries were significantly more susceptible to both Aspergillus colonization (5 to 15 times more susceptible) and OTA and FB2 contamination (2 to 9 times more susceptible) than were SS berries. The highest toxin concentration was detected in inoculated PM berries both for OTA (9 ng/g) at 20°C and for FB2 (687 ng/g) at 30°C. In inoculated SS and PM berries, although malondialdehyde and hydrogen peroxide concentrations did not increase, the two black Aspergillus species caused a significant decrease in ascorbate content, thus inducing a pro-oxidant effect. These results indicate that grape berries affected by PM are more susceptible to black Aspergillus growth and to production and/or accumulation of FB2 and OTA. Thus, preventive control of E. necator on grape berries could reduce the mycotoxicological risk from black Aspergillus infection.

Changes in lignin biosynthesis and monomer composition in response to benzothiadiazole and root-knot nematode Meloidogyne incognita infection in tomato

Benzothiadiazole (BTH) acts as a priming agent in plant defence leading to a reduction in penetration and development of the root-knot nematode Meloidogyne incognita in susceptible tomato roots. Changes in lignin biosynthesis in the susceptible tomato cv. Roma following nematode infection and/or BTH treatment were investigated in comparison to the resistant cv. Rossol. Both untreated and BTH-treated susceptible infected roots (galls) showed an increased level of expression of lignin synthesis-related genes (PAL, C4H, HCT and F5H) at early times during infection (2-4 days post inoculation). Peroxidase (soluble and cell-wall bound, POX) enzyme activities increased after inoculation with M. incognita and the priming effect of BTH treatment was evident at later stages of infection (7 days post inoculation). As expected, the induction of PAL and POXs and lignin synthesis-related genes was faster and greater in resistant roots after infection. Histochemical analysis revealed accumulation of higher lignin levels at later infection stages in BTH-treated galls compared to untreated ones. Furthermore, the monomer composition of lignin indicated a different composition in guaiacyl (G) and syringyl (S) units in BTH-treated galls compared to untreated galls. The increase in G units made G/S ratio similar to that in the resistant genotype. Overall, lignin played a critical role in tomato defence to M. incognita in response to BTH.