No abstract

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.

No abstract

In September 2008, a new disease was noticed in eastern Sicily, Italy in a 2-year-old regrafted citrus orchard with approximately 1,500 plants of sweet orange (Citrus sinensis (L.) Osbeck cv. Tarocco Scirè) on sour orange rootstock. Symptoms on the sweet orange scion consisted of blight of vigorously growing shoots and a sooty canker on shoots and rootstock trunks, resulting in shoot dieback to the cankered area. Masses of black fungal spores appeared under the bark and on the canker surface. Abundant gummosis was frequently associated with the affected tissues. Of the 1,500 plants surveyed, 12% were infected. A Scytalidium-like fungus was isolated consistently from symptomatic tissues on 2% potato dextrose agar (PDA). Conidia were ellipsoid to ovoid, hyaline, with an acutely rounded apex, truncate base, initially aseptate, becoming brown and two-septate at maturity, (10-) 12 to 13 (-14) × (4-) 5 (-6) μm. Mycelium was branched with septate, brown hyphae that disarticulated into 0- to 1-septate phragmospores (toruloid state). Genomic DNA was extracted from mycelia of single-conidial isolates cultivated on malt extract agar. Primers V9G and ITS4 were used to amplify the nuclear rRNA operon spanning the 3′ end of 18S rRNA gene, the internal transcribed spacers, the 5.8S rRNA gene, and a part of the 5′ end of the 28S rRNA gene (1,2). Both PCR primers were used to sequence directly the entire amplicon. DNA sequences of two isolates (CBS 124887 and 124888) were deposited in GenBank (Accession Nos. GQ330902 and GQ330903, respectively). These sequences were 100% identical in more than 545 nt to GenBank Accession Nos. AY213688 and FJ648577. On the basis of morphological characters and molecular data, the fungal isolates were identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers (2,3). Pathogenicity tests were conducted on five 2-year-old potted plants of sweet orange cv. Tarocco Scirè and lemon cv. Femminello Zagara Bianca, both grafted on sour orange. Eight 5-cm mycelial plugs of a single-conidial isolate were placed in wounds made with a sterile blade in the inner bark of plant stems and branches. Inoculation wounds were wrapped with Parafilm. The same number of plants inoculated with sterile PDA plugs served as controls. Inoculated plants were maintained in a growth chamber at 25 ± 1°C and 90 to 95% relative humidity. After 2 weeks, all inoculated plants developed gummosis originating from the inoculation point. Shoot blight and death of the entire plant were observed within 6 months on all inoculated plants. No differences were observed among the two citrus species. Control plants remained healthy. N. dimidiatum was reisolated from the infected plants and identified as described. To our knowledge, this is the first record of a disease caused by N. dimidiatum on citrus in Italy. The pathogen has been previously observed to infect freeze-damaged limbs of citrus in California, inducing a disease named Hendersonula branch wilt (4). This pathogen on citrus is important mainly as a wound-invading pathogen, therefore posing a serious threat to regrafted citrus orchards. References: (1) R. Cheewangkoon et al. Persoonia 21:77, 2008. (2) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (3) E. Punithalingam and J. M. Waterston. No 274 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1970. (4) J. O. Whiteside. Page 29 in: Compendium of Citrus Diseases. The American Phytopathological Society, St. Paul, MN, 1988.

During the period from 2010 to 2013 preharvest symptoms were detected on different cultivars of sweet orange in six orchards in Catania, Siracusa and Enna provinces, Southern Italy. A total of 56 monosporic fungal isolates were obtained, and among these, 44 were identified as Colletotrichum gloeosporioides and 12 as C. karstii through morphological and molecular analysis. PCR with primers ITS1 and ITS4, primers TubGF1 and TubGR specific for β‐tubulin gene, primers GDF‐GDR, specific for Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) gene, were used to confirm the identification of Colletotrichum isolates from citrus. The ITS1‐5.8S‐ITS2 region, a portion of approximately 500 bp of β‐tubulin gene and a fragment of 220 bp of GAPDH gene of the isolates were sequenced and analysed with the BLASTn program. Koch's postulates were fulfilled by pathogenicity tests carried out on fruit of ‘Tarocco Scirè’ and ‘Tarocco Nucellare’ with representative isolates of C. gloeosporioides and C. karstii. Field surveys and pathogenicity tests revealed significant differences in fruit susceptibility between ‘Tarocco Scirè’ and ‘Tarocco Nucellare’ and in virulence between the fungal species. To our knowledge, this is the first report on the emergence of Colletotrichum spp. causing anthracnose in preharvest conditions.

The biocontrol properties of the endophyte Pseudomonas synxantha DLS65 were tested in vitro and in vivo against Monilinia fructicola and Monilinia fructigena, causal agents of postharvest brown rot of stone fruit. P. synxantha cells significantly reduced the mycelial growth of both pathogens on Potato Dextrose Agar (PDA), and strongly inhibited the Monilinia fructicola growth on Peach Extract Agar (PEA). Cell-free culture filtrates inhibited the pathogens on PDA and PEA to lesser extent. The production of volatile organic compounds (VOCs), with in vitro inhibitory effects on mycelial growth, was also observed. P. synxantha significantly reduced brown rot incidence and severity on peach fruit artificially inoculated with M. fructicola after 5 d at 25°C. Moreover, P. synxantha more significantly reduced incidence and severity after 10 d at 10°C and after 20 d in cold storage at 0°C in comparison to control fruit, even if its activity was never comparable to that of the synthetic fungicide Scholar ® (fludioxonil). Similarly, P. synxantha exhibited an excellent antagonistic activity against M. fructigena on fruit at 10 and at 0°C, and a weak biocontrol activity at 25°C. Competition for nutrients and space, production of diffusible toxic metabolites and VOCs may play a role in the antagonism of P. synxantha toward M. fructicola and M. fructigena, especially at the lowest temperatures of storage. For that reason, this strain of P. synxantha could be suggested as active ingredient for the setting up of bioformulates against Monilinia species representing a limiting factor for stone fruit production.