Volatile profiles of healthy and aflatoxin contaminated pistachios.

The volatile profile of four different groups of dried pistachios namely: H: healthy, NC: naturally contaminated with aflatoxin, AC: artificially contaminated with aflatoxigenic strains of the fungi Aspergillus flavus and ANT: artificially contaminated with non-toxigenic strains of the fungi A. flavus, was determined. The volatiles were isolated by the HS-SPME method and determined by GC-FID and GC-MS, whereas aflatoxin by HPLC. Seventy two volatile compounds were identified almost equally distributed among the above four studied groups. The predominant chemical compounds were monoterpenes, alcohols, ketones, aldehydes, esters and hydrocarbons. The monoterpenes, mainly determined as α-pinene and α-terpinolene were detected in all samples. Even though the general volatile profile was similar among groups, some differences were detected between healthy and contaminated groups of samples. When some key volatiles such as eight-carbon and seven-carbon alcohols and aldehydes were used along with the species-specific sesquiterpenes and the other terpenes detected, a correct classification was obtained in H, NC, AC and ANT groups, as was demonstrated by cluster and discriminant analyses. This evidence provides a potential tool for distinguishing contaminated samples on the basis of characteristic volatile patterns.

First Record of Phytophthora nicotianae Causing Leaf Blight on Rhododendron in Greece.

Species of Rhododendron and resulting hybrids are very important hosts of the quarantine pathogen Phytophthora ramorum, since they play a major role in the spread of the pathogen in Europe. However, many other Phytophthora species infect these hosts, causing similar symptoms. Widmer (4) listed 17 Phytophthora species as foliar pathogens of rhododendron in the United States. A survey was conducted in Greece in October 2009, in which potted plants of Rhododendron spp. were inspected for symptoms of necrotic lesions on leaves and buds caused by P. ramorum. Symptomatic plants were observed in one of the nurseries inspected in the Triphylia Region in southwestern Peloponnese. Isolations from symptomatic leaves on PARBhy-V8 selective agar medium (1) yielded Phytophthora isolates. Colonies on V8 juice agar appeared white and cottony, with a radial growth of 4.2 to 4.6 mm per day at 28°C with a maximum growth temperature of 36°C. Sporangia were produced abundantly on the medium surface and in water; the sporangia were broadly ovoid and papillate and 35 to 50 × 25 to 35 µm. Chlamydospores, 25 to 40 µm in diameter, were observed in 2-week-old cultures, while no sexual structures were observed. Three of the isolates examined were identified as P. nicotianae B. de Haan on the basis of morphological and physiological features (3,4). Genomic DNA was extracted from pure cultures of an isolate and the internal transcribed spacer (ITS) region was amplified using the ITS4/5 primer pair. Sequence analyses by BLAST indicated that the isolates were most similar to P. nicotianae (GenBank Accession No. AJ 854295.1) with sequence identity values of 99%. One of the isolates was deposited in the culture collection of the University of Athens (ATHUM 6519). Detached wounded leaves of Rhododendron hybrid cv. Red Jack were inoculated with agar plugs. Necrotic lesions, similar to those observed in the nursery, appeared on the inoculated leaves after 7 days of incubation at 26°C, while no symptoms developed on control leaves inoculated with sterile agar plugs. The pathogen was consistently reisolated from infected leaves, but not from the controls. P. nicotianae, being a thermophilic species, is the most common Phytophthora species in Greece, reported on more than 30 plant species (2). This pathogen has been reported on Rhododendron spp. in the United States (3,4), but to our knowledge, there was no record of this pathogen on these hosts as yet in Europe and this is the first published report of the pathogen on Rhododendron in Greece. References: (1) A. Belisario et al. Plant Dis. 87:101, 2003. (2) K. Elena. Technical Bulletin No 13. Benaki Phytopathological Institute. Athens, Greece (in Greek), 1999. (3) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (4) T. L. Widmer. Online publication. doi: 10.1094/PHP-2010-0317-01-RS, Plant Health Progress, 2010.

First Report of Phytophthora ramorum on Rhododendron in Greece.

In April 2010, during a survey conducted in Fthiotis Prefecture of central Greece, symptoms of stem necrosis and leaf lesions were observed on two container-grown plants of Rhododendron, hybrid 'Kate Waterer' in a nursery. From symptomatic leaves, a Phytophthora species was isolated on PARPH-V8 selective agar medium (2) with typical morphological characters of Phytophthora ramorum S. Werres & A.W.A.M. de Cock (4). The whole block of plants was under probation until molecular verification of the pathogen was completed. The nursery was reexamined 6 weeks after the first encounter, whereas spread of the pathogen was noticed to neighboring plants in the same block; five more Rhododendron plants with similar symptoms were recorded while one of the originally infected plants was dead. Isolates of Phytophthora with similar morphology were obtained from symptomatic leaves of three new plants as well as from the potting mix of a severely infected plant that was baited in a Rhododendron leaf assay (2). All Rhododendron plants in the block belonged to the same consignment imported from Belgium and covered by a phytosanitary plant passport. Colonies on 10% clarified V8 juice agar appeared with coralloid, coenocytic mycelium with radial growth at 1.7 mm per day at 20°C and maximum temperature 26 to 27°C. Propagules characteristic of P. ramorum, including semipapillate, caducous, sporangia measuring 35 to 55 × 15 to 30 µm (1.9 length/width ratio) and large chlamydospores (45 to 80 µm), were observed on V8 agar. One isolate was confirmed as P. ramorum by sequence analysis of the internal transcribed spacer region of rDNA and was deposited in the culture collection of the University of Athens (ATHUM 6522). Comparison of amplicon sequences (using ITS4/5 primer pair) of approximately 875 bp long was carried out using MEGABLAST search for highly similar sequences. Alignment data revealed the highest and most significant homology to P. ramorum (GenBank Accession No. AY594198.1) at 99%. Pathogenicity tests were carried out using detached leaves of Rhododendron hybrid 'Red Jack' and Arbutus unedo L., which were slightly wounded and inoculated with mycelium agar plugs (3). Necrotic lesions appeared on the inoculated leaves of both plant species 10 days after incubation at 20°C, while no symptoms developed on control leaves inoculated with sterile agar plugs. P. ramorum was consistently reisolated from artificially infected leaves of both plant species. Following confirmation of pathogen presence, eradication measures were applied in the nursery. The adverse weather conditions encountered in summer, with temperatures very often above 35°C, are expected to favor pathogen eradication. However, not all plants of the same consignment imported from Belgium were traced and it is possible that other infected plants have been sold in other areas of Greece. So far, P. ramorum had been reported in 21 other European countries; Serbia is the nearest country where the pathogen was detected (1). To our knowledge, this is the first report of P. ramorum in Greece. References: (1) A. Bulajic et al. Plant Dis. 94:703, 2010. (2) E. J. Fichtner et al. Phytopathology 97:1366, 2007. (3) R. G. Linderman et al Online publication. doi:10.1094/PHP-2007-0917-01-RS. Plant Health Progress, 2007. (4) S. Werres et al. Mycol. Res. 105:1155, 2001.

First Report of Fomitiporia punctata Infecting Kiwifruit.

During 2000 and 2001, a wood discoloration and decay of 10- to 12-year-old kiwifruit vines (Actinidia deliciosa (Chev.) Liang & Ferguson) was observed in vineyards in all major kiwifruit-growing areas of Greece. Leaves became chlorotic or necrotic, dry, and abscised prematurely. Fruits dropped before reaching full maturity. Necrosis of shoots and twigs progressed from the shoot tips toward the trunk. If the infected twigs were pruned early, the vine occasionally produced new shoots. Cross sections of infected trunks and shoots revealed a light-colored soft rot in the center, surrounded by brown, hard, necrotic wood resembling the esca disease of grapevine, caused by Fomitiporia punctata. From the white rotted areas, a fungus was isolated that formed a dense aerial mycelium with cream-yellow to light-brown colonies on potato dextrose agar (PDA). The fungus was identified as F. punctata (Fr.) Murrill by M. Fischer, Weinbauinstitut, Freiburg-Germany, (personal communication). Pathogenicity tests were performed by artificially inoculating 2-year-old healthy kiwifruit (cv. Hayward) plants. The inoculum consisted of a 7-mm diameter mycelial plug taken from the margin of a 10-day-old culture of F. punctata growing on PDA. A portion of the bark was partially raised from the trunk, and the inoculum was inserted under the bark. The bark was tied with adhesive tape to keep the inoculum in place and sealed with Vaseline to prevent dehydration. Ten plants were inoculated while 10 were used as nonincoculated controls. Wilting of lower leaves was first observed 15 days after inoculation. In longitudinal sections, a brown discoloration on the stem wood was observed 46 days after inoculation in all inoculated vines, while control plants remained healthy. However, the same fungus was isolated from only one infected plant. Low frequency in reisolating F. punctata is often mentioned in the literature (3). Although esca on kiwifruit has been reported in Italy, the fungus initially was identified as Phellinus conchatus and subsequently as F. punctata (1,2). To our knowledge, this is the first published report of F. punctata causing wood decay of kiwifruit. References: (1) F. Calzarano et al. Inf. Fitopatol. 49:12, 1999. (2) S. Di Marco et al. N. Z. J. Crop Hortic. Sci. 28:69, 2000. (3) L. Sparapano et al. Phytopathol. Mediterr. 39:46, 2000.

Pseudorecombination between infectious cloned DNA components of tomato mottle and bean dwarf mosaic geminiviruses.

A newly described whitefly-transmitted geminivirus infecting tomato plants in Florida induces yellow mottling symptoms on leaves, and stunted and distorted growth. The DNA-A and DNA-B components were cloned from extracts of field-infected tomato tissue; excised monomers or uncut tandem dimers of these clones were infectious when co-inoculated on to Nicotiana benthamiana by rub-inoculation. Tomato plants inoculated directly with the DNA-A and DNA-B dimers, or indirectly by sap or graft transmission from N. benthamiana plants previously infected with the dimers, developed symptoms similar to those observed in field-infected plants. This tomato geminivirus is different from previously characterized geminiviruses, and has been named tomato mottle geminivirus (ToMoV). DNA sequence comparisons revealed that ToMoV is closely related to bean dwarf mosaic geminivirus (BDMV) and abutilon mosaic geminivirus. Infectious pseudorecombinants were made by exchanging the cloned infectious DNA components of ToMoV and BDMV and inoculating N. benthamiana plants. The presence of the inoculated DNA components in systemically infected plants was confirmed by characterization of DNA-A and DNA-B fragments amplified by the polymerase chain reaction. This is the first report of pseudorecombination between two distinct geminiviruses. The implications of this finding in geminivirus evolution are discussed.