First Report of Powdery Mildew Caused by Golovinomyces orontii (Erysiphe orontii) on Lamium galeobdolon in Italy.

Lamium galeobdolon L. (Labiatae) is a common ornamental species that grows in shade areas and often used as a ground cover in gardens. During the summer of 2006, severe outbreaks of a previously unknown powdery mildew were observed on all Lamium spp. plants in some gardens near Biella (northern Italy). Both surfaces of the leaves of affected plants were covered with dense, white mycelia and conidia. As the disease progressed, infected leaves turned yellow and died. Mycelia and conidia also were observed on stems and flowers. Conidia were hyaline, ellipsoid, borne in short chains (with a maximum of five conidia per chain), and measured 29 to 37 × 16 to 20 µm (average 33 × 18 µm). Conidiophores, 91 to 104 µm (average 96 µm) long, showed the foot cell measuring 28 to 49 × 9 to 11 µm (average 38 × 10 µm), followed by three shorter cells measuring 14 to 26 × 9 to 15 µm (average 21 × 11 µm). Fibrosin bodies were absent. Chasmothecia were not observed in the collected samples. The internal transcribed spacer region (ITS) of rDNA was amplified using the primers ITS4/ITS6 (4) and sequenced. BLASTn analysis (1) of the 436 bp obtained showed an E-value of 0.0 with Golovinomyces orontii (Erysiphe orontii.) (3). The nucleotide sequence has been assigned GenBank Accession No. EF 121871. Inoculations were made by gently pressing diseased leaves onto leaves of five healthy L. galeobdolon plants. Five noninoculated plants served as controls. Inoculated and noninoculated plants were maintained in a greenhouse at temperatures between 15 and 28°C. After 10 days, typical powdery mildew colonies developed on inoculated plants. Noninoculated plants did not show symptoms. The pathogenicity test was carried out twice. To our knowledge, this is the first report of the presence of powdery mildew on L. galeobdolon caused by G. orontii in Italy. Blumer (2) was able to reproduce powdery mildew symptoms on L. galeobdolon using populations from cucumber, while Braun (3) reported L. galeobdolon as a possible host of E. orontii. Herbarium specimens of this disease are available at AGROINNOVA Collection, University of Torino, Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) S. Blumer. Ber. Schweiz. Bot. Ges. 62:384, 1952. (3) U. Braun. A Monograph of the Erysiphaceae (Powdery Mildews). Cramer, Berlin, GDR, 1987. (4) D. E. L. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997.

First Report of Phytophthora citrophthora on Penstemon barbatus in Italy.

Penstemon barbatus (Cav.) Roth (synonym Chelone barbata), used in parks and gardens and sometimes grown in pots, is a plant belonging to the Scrophulariaceae family. During the summers of 2004 and 2005, symptoms of a root rot were observed in some private gardens located in Biella Province (northern Italy). The first symptoms resulted in stunting, leaf discoloration followed by wilt, root and crown rot, and eventually, plant death. The diseased tissue was disinfested for 1 min in 1% NaOCl and plated on a semiselective medium for Oomycetes (4). The microorganism consistently isolated from infected tissues, grown on V8 agar at 22°C, produced hyphae with a diameter ranging from 4.7 to 5.2 µm. Sporangia were papillate, hyaline, measuring 43.3 to 54.4 × 26.7 to 27.7 µm (average 47.8 × 27.4 µm). The papilla measured from 8.8 to 10.9 µm. These characteristics were indicative of a Phytophthora species. The ITS region (internal transcribed spacer) of rDNA was amplified using primers ITS4/ITS6 (3) and sequenced. BLASTn analysis (1) of the 800 bp obtained showed a 100% homology with Phytophthora citrophthora (R. & E. Sm.) Leonian. The nucleotide sequence has been assigned GenBank Accession No. DQ384611. For pathogenicity tests, the inoculum of P. citrophthora was prepared by growing the pathogen on autoclaved wheat and hemp kernels (2:1) at 25°C for 20 days. Healthy plants of P. barbatus cv. Nano Rondo, 6 months old, were grown in 3-liter pots (one plant per pot) using a steam disinfested substrate (peat/pomix/pine bark/clay 5:2:2:1) in which 200 g of kernels per liter of substrate were mixed. Noninoculated plants served as control treatments. Three replicates were used. Plants were maintained at 15 to 20°C in a glasshouse. The first symptoms, similar to those observed in the gardens, developed 21 days after inoculation, and P. citrophthora was consistently reisolated from infected plants. Noninoculated plants remained healthy. The pathogenicity test was carried out twice with similar results. A nonspecified root and crown rot of Penstemon spp. has been reported in the United States. (2). To our knowledge, this is the first report of P. citrophthora on P. barbatus in Italy as well as in Europe. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997 (2) F. E. Brooks and D. M. Ferrin. Plant Dis. 79:212, 1995. (3) D. E. L. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997. (4) H. Masago et al. Phytopathology 67:425, 1977.

First Report of Powdery Mildew Caused by Erysiphe (Golovinomyces) orontii on Veronica spicata in Italy.

Veronica spicata (spike speedwell) is a perennial garden species belonging to the family Scrophulariaceae. During the summer through fall of 2004 and 2005, severe outbreaks of a previously unknown powdery mildew were observed in several gardens near Biella (northern Italy). Upper surfaces of leaves were covered with a white mycelium and conidia, and as the disease progressed, infected leaves turned yellow and died. Very rarely was the mycelium observed on the lower surface of leaves or on petioles and flowers. Foot cell was cylindric and measured 19.2 to 25.7 × 10.8 to 14.3 µm (average 21.9 × 12.0 µm). Conidia were hyaline, ellipsoid, brought in short chains (three conidia per chain), and measured 22.2 to 40.8 × 13.6 to 21.6 µm (average 30.1 × 17.0 µm). Conidiophores measured 45.5 to 74.0 × 10.4 to 11.0 µm (average 59.4 × 10.6 µm). Fibrosin bodies were absent. Cleistothecia were never observed on the samples collected. The ITS region (internal transcribed spacer) of rDNA was amplified using the primers ITS4/ITS6 (3) and sequenced. BLASTn analysis (1) of the 504 bp obtained showed an E-value of 0.0 with Erysiphe (Golovinomyces) orontii (2). The nucleotide sequence has been assigned GenBank Accession No. DQ386696. Pathogenicity was confirmed by gently pressing diseased leaves onto leaves of five healthy Veronica spicata plants. Five noninoculated plants served as controls. Inoculated and noninoculated plants were maintained in a greenhouse where temperatures ranged between 15 and 28°C. After 15 days, typical powdery mildew symptoms developed on inoculated plants. Noninoculated plants did not show symptoms. The pathogenicity test was carried out twice. To our knowledge, this is the first report of the presence of powdery mildew on V. spicata in Italy. Sphaerotheca fuliginea has been reported as the causal agent of powdery mildew on V. spicata (4). Specimens of this disease are available at DIVAPRA Collection, University of Torino. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) U. Braun. Nova Hedwigia 89:166, 1987. (3) D. E. L. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997. (4) B. Ing. Mycologist 4:125, 1990.

First Report of Leaf Spot Caused by Phoma exigua on Hydrangea macrophylla in Italy.

Hydrangea macrophylla is grown in Italy as a potted plant and also for landscaping. During the fall of 2005 in a nursery located in Lazio (central Italy), a severe foliar disease was observed on 12-month-old potted plants of cv. Hanabi. Small necrotic spots surrounded by chlorotic haloes were observed on the upper side of infected leaves. At temperatures near 20°C and relative humidity ranging between 80 to 90%, spots enlarged to form round areas 2 to 7 cm in diameter that were well defined by a brown margin. Severely infected leaves became chlorotic and abscised. Heavily affected plants were defoliated. Infected plants rarely died, but the presence of lesions on mature plants decreased aesthetic quality and subsequent market value. The disease occurred on 30% of the plants in one nursery. Stems and flowers were not affected by the disease. From infected leaves, a fungus was consistently isolated on potato dextrose agar with 25 mg/liter of streptomycin added. The fungus was grown on leaf extract agar, 30 g of leaves per liter, and maintained at 22°C (12 h of light and 12 h of dark). After 30 days, black pycnidia 275 to 255 µm in diameter developed, releasing conidia that were hyaline, elliptical, nonseptate, and measuring 4.6 to 7.6 (average 6.0) × 1.4 to 4.2 (average 2.6) µm. On oatmeal agar, the addition of a drop of concentrated NaOH caused a positive reaction, turning the medium red (2). On the basis of its morphological characteristics, the fungus was identified as a Phoma sp. The ITS (internal transcribed spacer) region of rDNA was amplified using the primers ITS4/ITS6 (3) and sequenced. BLASTn analysis (1) of the 560 bp obtained showing an E-value of 0.0 with Phoma exigua. The nucleotide sequence has been assigned GenBank Accession No. DQ384612. Pathogenicity tests were performed by spraying leaves of healthy 6-month-old potted H. macrophylla (cvs. Hanabi and Zaffiro) plants with a spore and mycelial suspension (105 CFU/ml). Plants without inoculation served as controls. Five plants were used for each treatment. Plants were covered with plastic bags for 5 days after inoculation and kept in a growth chamber at 20°C with relative humidity at 80 to 90%. The first lesions developed on leaves of cv. Hanabi 12 days after inoculation, while control plants remained healthy. Lesions did not develop on inoculated cv. Zaffiro plants. The fungus was consistently reisolated from the lesions of cv. Hanabi. The pathogenicity test was carried out twice. The presence of P. exigua on H. macrophylla has been reported in the United States (4). In Italy, the disease can be found in a limited area. References: (1) S. F. Altschud et al. Nucleic Acids Res. 25:3389, 1997. (2) G. H. Boerema and L. H. Howeler. Persoonia 5:15, 1967. (3) D. E. L. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997. (4) M. L. Daughtrey et al. Page 26 in: Compendium of Flowering Potted Plant Diseases. The American Phytopathological Society. St. Paul, MN, 1995.

Detection and identification of bacterial endosymbionts in arbuscular mycorrhizal fungi belonging to the family Gigasporaceae.

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.

Nitrogen fixation genes in an endosymbiotic Burkholderia strain.

In this paper we report the identification and characterization of a DNA region containing putative nif genes and belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita. A genomic library of total DNA extracted from the fungal spores was also representative of the bacterial genome and was used to investigate the prokaryotic genome. Screening of the library with Azospirillum brasilense nifHDK genes as the prokaryotic probes led to the identification of a 6,413-bp region. Analysis revealed three open reading frames encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of the component I and with component II (NifH) of nitrogenase from different diazotrophs. The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs. PCR experiments with primers designed on the Burkholderia nifHDK genes and Southern blot analysis demonstrate that they actually belong to the genome of the G. margarita endosymbiont. They offer, therefore, the first sequence for the nif operon described for Burkholderia. Reverse transcriptase PCR experiments with primers designed on the Burkholderia nifH and nifD genes and performed on total RNA extracted from spores demonstrate that the gene expression was limited to the germination phase. A phylogenetic analysis performed on the available nifK sequences placed the endosymbiotic Burkholderia close to A. brasilense.

An obligately endosymbiotic mycorrhizal fungus itself harbors obligately intracellular bacteria.

Arbuscular-mycorrhizal fungi are obligate endosymbionts that colonize the roots of almost 80% of land plants. This paper describes the employment of a combined morphological and molecular approach to demonstrate that the cytoplasm of the arbuscular-mycorrhizal fungus Gigaspora margarita harbors a further bacterial endosymbiont. Intracytoplasmic bacterium-like organisms (BLOs) were detected ultrastructurally in its spores and germinating and symbiotic mycelia. Morphological observations with a fluorescent stain revealed about 250,000 live bacteria inside each spore. The sequence for the small-subunit rRNA gene obtained for the BLOs from the spores was compared with those for representatives of the eubacterial lineages. Molecular phylogenetic analysis unambiguously showed that the endosymbiont of G. margarita was an rRNA group II pseudomanad (genus Burkholderia). PCR assays with specifically designed oligonucleotides were used to check that the sequence came from the BLOs. Successful amplification was obtained when templates from both the spores and the symbiotic mycelia were used. A band of the expected length was also obtained from spores of a Scutellospora sp. No bands were given by the negative controls. These findings indicate that mycorrhizal systems can include plant, fungal, and bacterial cells.