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Branch cankers and stem-end rot are two of the most important threats to avocado production. During the autumn of 2013, sampling was conducted in the main avocado growing area in eastern Sicily to study the occurrence and establish the causal agents of branch canker and stem-end rot. A total of 94 fungal isolates, recovered from four avocado orchards, were identified by morphological characterisation, DNA sequencing and phylogenetic analyses as belonging to the genera Colletotrichum, Neofusicoccum or Diaporthe. The majority of the isolates were identified as Neofusicoccum parvum (70.2 %), with the remaining isolates being Colletotrichum gloeosporioides or C. fructicola (16 %), and Diaporthe foeniculacea or D. sterilis (13.8 %), respectively. Pathogenicity tests showed N. parvum was the most virulent species (P = 0.05), whereas Diaporthe isolates were the least so. An intermediate virulence was observed for C. gloeosporioides and C. fructicola, which were associated only with stem-end rot of fruit. Regarding cultivar susceptibility of fruit to these pathogens, 'Hass' was more susceptible to infection by C. fructicola and D. foeniculacea compared with 'Bacon' whereas no significant differences were detected for the remaining pathogens. To our knowledge, this is the first account of the pathogens causing branch canker and stem-end rot of avocado in Italy, and the first studies comparing the relative virulence of each species involved.

Antifungal genes from micro-organisms were inserted into the genome of a number of plant species, representing a promising strategy for conferring genetic disease resistance against a broad range of plant pathogenic fungi. In the present study, the chit42 gene from Trichoderma harzianum (codifying the antifungal protein endochitinase) was introduced into the ÔFemminello siracusanoÕ lemon by Agrobacterium tumefaciens, in order to regenerate transgenic plants resistant to fungal disease. Three polymerase chain reaction (PCR)-positive clones were obtained. Southern blot confirmed the integration of the transgene in the lemon genome and revealed that one or two copies had been inserted. Reverse transcriptase-PCR, Northern blot and Western analysis were performed and the results confirmed the expression of the inserted gene. The transgenic clones were tested in vitro and in vivo for disease resistance. Conidia germination and fungal growth of Phoma tracheiphila were strongly inhibited in vitro by the transgenic foliar proteins, while no effects were observed with the controls. Disease resistance assays were performed in vivo with Botrytis cinerea, the causal agent of grey mould in fruit. Transgenic lemon plants, inoculated with lemon petals infected by a single-conidial isolate of B. cinerea, showed significantly less lesion development than the controls. On the whole, the results indicate that the transformation with the antifungal endochitinase gene may represent a strategy for disease control in citrus crops.

Constitutive over-expression of antifungal genes from microorganisms involved in plant defence mechanisms represents a promising strategy for conferring genetic resistance against a broad range of plant pathogenic fungi. In the present work, two transgenic lemon clones with the chit42 gene from Trichoderma harzianum were tested for resistance to fungal disease and expression level of defence-related genes was evaluated. Different resistance-related processes, such as production of reactive oxygen species (ROS), systemic acquired resistance (SAR) and induced systemic resistance (ISR), were monitored in transgenic and wild type lemon clones inoculated with Botrytis cinerea, the causal agent of grey mould in citrus. Expression of genes that encode gluthatione peroxidase (GPX), a producer of ROS, chitinases, glucanases (SAR), PAL, HPL, and AOS (ISR) was measured by quantitative PCR during the first 24 h after leaf inoculation. Leaves of transgenic lemon plants inoculated with B. cinerea showed significantly less lesion development than wild type leaves. Tissues from detached leaves of different transgenic lemon clones showed a significant correlation between resistance and transgene expression. On the other hand, the over-expression of the transgenic fungal gene enhanced by two-three folds transcript levels of genes associated with enhanced ROS production and ISR establishment, while the expression of native chitinase and glucanase genes involved in SAR was down-regulated.

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