Efficacy of Biofumigation with Brassica carinata Commercial Pellets (BioFence) to Control Vegetative and Reproductive Structures of Phytophthora cinnamomi.

The efficacy of biofumigation with Brassica carinata pellets (BioFence) to control vegetative and reproductive structures of Phytophthora cinnamomi was investigated in vitro at different doses and temperatures. Biofumigation was effective in inhibiting mycelial growth (culture diameter) and chlamydospore and zoospore germination, and was lethal at 24 mg of pellet per plate (approximately 0.4 mg/liter). The 50% effective concentration values showed that efficacy of B. carinata pellets in inhibiting or killing the vegetative and reproductive structures of P. cinnamomi was maximum at 15°C and decreased as temperature rose to 25°C. However, the fungicide effect was independent of the temperature. In vivo biofumigation of Quercus cerris seedlings with BioFence confirmed efficacy by reducing the inoculum density (CFU/g) of P. cinnamomi, thus protecting the host from root infection. The use of BioFence provides an alternative to synthetic pesticides to control P. cinnamomi within disease management programs in agroforestry systems.

Pathogenicity of Phytophthora lateralis Lineages on Different Selections of Chamaecyparis lawsoniana.

Phytophthora lateralis, the cause of Chamaecyparis lawsoniana root disease, was introduced in North America about 1920, and has since killed trees along roads and streams throughout the tree's range. Recent results suggest an Asian origin for this oomycete and four genetic lineages were identified. This raised questions for the genetic exapted resistance demonstrated in 1989 within the wild population of C. lawsoniana but with only one P. lateralis lineage. The main goal of the present research was to test the durability of the demonstrated resistance and to compare the pathogenicity of isolates representing the four lineages. No breakdown of resistance was observed in five separate tests using different inoculation techniques, resistant and susceptible C. lawsoniana trees, and seedling families. Differences in mortality and lesion length were observed between the lineages. The higher aggressiveness of isolates of the TWJ and PNW lineages and the lower aggressiveness of the TWK lineage are discussed in view of the hypotheses on the history of spread and evolutionary history of the P. lateralis lineages.

Biogeographical patterns and determinants of invasion by forest pathogens in Europe.

A large database of invasive forest pathogens (IFPs) was developed to investigate the patterns and determinants of invasion in Europe. Detailed taxonomic and biological information on the invasive species was combined with country-specific data on land use, climate, and the time since invasion to identify the determinants of invasiveness, and to differentiate the class of environments which share territorial and climate features associated with a susceptibility to invasion. IFPs increased exponentially in the last four decades. Until 1919, IFPs already present moved across Europe. Then, new IFPs were introduced mainly from North America, and recently from Asia. Hybrid pathogens also appeared. Countries with a wider range of environments, higher human impact or international trade hosted more IFPs. Rainfall influenced the diffusion rates. Environmental conditions of the new and original ranges and systematic and ecological attributes affected invasiveness. Further spread of established IFPs is expected in countries that have experienced commercial isolation in the recent past. Densely populated countries with high environmental diversity may be the weakest links in attempts to prevent new arrivals. Tight coordination of actions against new arrivals is needed. Eradication seems impossible, and prevention seems the only reliable measure, although this will be difficult in the face of global mobility.

First Report of Phytophthora × pelgrandis Causing Root Rot and Lower Stem Necrosis of Common Box, Lavender and Port-Orford-Cedar in Hungary.

In 2008 and 2009, necrotic bark lesions at the root collar and lower stem associated with root rot, reduced growth, and wilting were observed on container-grown common box (Buxus sempervirens L.), lavender (Lavandula angustifolia Mill. 'Hidcote'), and Port-Orford-cedar (Chamaecyparis lawsoniana (A. Murray) Parl. 'Columnaris') in three ornamental nurseries in western Hungary. Number of affected plants ranged from approximately 100 (Port-Orford-cedar) to 250 (lavender). Isolations from necrotic root collars of each host plant species yielded four Phytophthora isolates developing uniform colonies on carrot agar with a maximum growth temperature of 35 to 36°C. The isolates were homothallic with smooth-walled oogonia (32.2 ± 2.3 to 35.9 ± 3.5 µm), aplerotic oospores (27.5 ± 1.8 to 32.1 ± 3.1 µm) and both amphigynous and paragynous antheridia, and produced chlamydospores (25.8 ± 3.9 to 29.1 ± 5.2 µm) and papillate sporangia (35.2 ± 2.5 to 43.5 ± 5.6 µm long and 27.6 ± 2.2 to 32.0 ± 3.8 µm wide), mostly obpyriform to nearly spherical or rarely distorted with two or three apices. In spring water, sporangia were both caducous with short pedicel and non-caducous. Multiplex ITS-PCR assay of DNA from all isolates, using primers specific for P. nicotianae (NICF1 and NICR2.1) and P. cactorum (CACTF1 and CACTR1) (1), amplified DNA fragments of the expected size for each Phytophthora species. In addition, isoenzyme analysis revealed a characteristic banding pattern of one heterodimer and two homodimer bands at both loci of the dimeric enzyme malate dehydrogenase. These bands comigrated with those of P. × pelgrandis (Gerlach et al.) (CBS 123385) and isolate PD 93/1339 (courtesy of W. A. Man in 't Veld), two natural hybrid strains of P. nicotianae and P. cactorum (2,3), proving that our four isolates can be referred to as this interspecific hybrid. Pathogenicity was tested on 1- or 3-year-old plants of the original host species and cultivars (for common box, cv. Faulkner was used). Cultures were grown for 4 to 6 weeks at 20°C on autoclaved millet grains moistened with V8 broth. Infested and uninfested grains were mixed with autoclaved soil in a ratio of 6% (w/v), and the mixes were used as potting media for transplanting five treated and five control plants per isolate, respectively. Plants were kept in a growth chamber (20°C, 70% RH, 12-h photoperiod). Pots were flooded for 24 h on the 1st and 21st day after transplanting. All plants in infested potting mix showed symptoms of wilt associated with basal stem and root necrosis, similar to those observed on the plants from the field, within 2 and 3 months on lavender and both common box or Port-Orford-cedar, respectively. Additionally, a reduction of root weight ranging from 35 to 68% compared to the control was recorded. Growth reduction was significant at P ≤ 0.019 according to Mann Whitney test. Control plants remained healthy. The same Phytophthora hybrid was reisolated solely from inoculated plants. In Europe, hybrid isolates of P. nicotianae × P. cactorum have been reported on several ornamental plants, including lavender, in the Netherlands and Germany (2,3). However, to our knowledge, this is the first report of this hybrid in Hungary and as a pathogen of common box and Port-Orford-cedar in the world. References: (1) P. J. M. Bonants et al. Phytopathology 90:867, 2000. (2) W. A. Man in 't Veld et al. Phytopathology 88:922, 1998. (3) H. I. Nirenberg et al. Mycologia 101:220, 2009.

Pyrosequencing as a tool for the detection of Phytophthora species: error rate and risk of false Molecular Operational Taxonomic Units.

AIMS: To evaluate the accuracy of pyrosequencing for the description of Phytophthora communities in terms of taxa identification and risk of assignment for false Molecular Operational Taxonomic Units (MOTUs). METHODS AND RESULTS: Pyrosequencing of Internal Transcribed Spacer 1 (ITS1) amplicons was used to describe the structure of a DNA mixture comprising eight Phytophthora spp. and Pythium vexans. Pyrosequencing resulted in 16 965 reads, detecting all species in the template DNA mixture. Reducing the ITS1 sequence identity threshold resulted in a decrease in numbers of unmatched reads but a concomitant increase in the numbers of false MOTUs. The total error rate was 0·63% and comprised mainly mismatches (0·25%) CONCLUSIONS: Pyrosequencing of ITS1 region is an efficient and accurate technique for the detection and identification of Phytophthora spp. in environmental samples. However, the risk of allocating false MOTUs, even when demonstrated to be low, may require additional validation with alternative detection methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytophthora spp. are considered among the most destructive groups of invasive plant pathogens, affecting thousands of cultivated and wild plants worldwide. Simultaneous early detection of Phytophthora complexes in environmental samples offers an unique opportunity for the interception of known and unknown species along pathways of introduction, along with the identification of these organisms in invaded environments.

First Report of Buxus rotundifolia Root and Collar Rot Caused by Phytophthora citrophthora in Italy.

Boxwood (Buxus spp.) includes several species of popular ornamental shrubs used in traditional and contemporary gardening. In March of 2008, a gradual and irreversible decline was observed on 6-year-old Buxus rotundifolia plants in a garden in central Italy. Of 150 boxwood plants, 70% were symptomatic, and among them, approximately 25% were completely wilted. Aboveground symptoms included stunting, necrotic bark lesions at the base of the stem, reduced growth, and leaf chlorosis. Leaves at first appeared light green, then turned yellow, bronze, or straw colored. Foliar symptoms were restricted to a few branches or extended to the whole crown. Immunological field tests (Pocket Diagnostic, CSL Diagnostics, Milan, Italy) on necrotic rootlets and bark tissues suggested the risk of the presence of Phytophthora spp. One species was consistently isolated on PARHP (2) from necrotic tissues of three symptomatic plants. Recovered isolates were heterothallic and produced aerial mycelium. Star-like compact growth was also observed on potato dextrose agar. Cultures on carrot agar submerged in saline solution (2) developed papillate, noncaducous, and sympodially branched sporangia of different shapes ranging from ovoid to obturbinate. Sporangia were 37.5 to 65.0 × 22.5 to 35.0 µm (average 49.0 × 31.0 µm); L/W ratio from 1.1 to 2.1 (average 1.6). On the basis of morphological features, these isolates belong to P. citrophthora (R.E. Sm. & E.H. Sm.) Leonian (2). The identity was confirmed by sequencing of the internal transcribed spacer region of the rDNA (NCBI Accession No. FJ874794). Inoculum for two isolates of P. citrophthora (3BO and 4BO) was produced for pathogenicity testing on 100 g of autoclaved millet grains moistened with 70 ml of V8 juice. Two, 2-liter aliquots of potting media were each amended with the infested grains (1:20 vol/vol) and five 3-year-old plants of B. rotundifolia per isolate were transplanted into the media. Plants were maintained in a growth chamber at 18 to 22°C, with relative humidity of 80%, and a 12-h photoperiod. Sterile millet grains were used to inoculate five boxwood plants for the control. After 2 months, control plants remained healthy while inoculated plants displayed root necroses and foliar symptoms similar to those observed in the field. The pathogen, consistently reisolated from roots of infected plants, reduced the root weight by an average of 25%. Root rot of boxwood plants is reported to be caused by P. citricola in Poland (4), P. parasitica in Virginia (3), and P. citrophthora in North Carolina (1). To our knowledge, this is the first report of P. citrophthora on B. rotundifolia in Europe. References: (1) Z. G. Abad et al. Plant Dis. 78:830, 1994. (2) D. C. Erwin and O. K. Ribeiro. Page 562 in: Phytophthora Diseases Worldwide. The American Phytopathological Society. St. Paul, MN, 1996. (3) M. A. Hansen. Plant Dis. Fact Sheets. 450-614W, 2000. (4) L. B. Orlikowski. J. Plant Prot. Res. 46:163, 2006.

First Report of Fusarium solani Causing Wilt of Olea europaea in Nepal.

Leaf drop, wilt, and mortality were observed in September of 2007 on approximately 10% of 1- to 2-year-old olive (Olea europaea cv. Leccino) plants shipped from Europe and growing in a nursery in the District of Makwampur, Nepal. Roots of symptomatic and asymptomatic plants were disinfected in 1% NaOCl, cut into 1 cm long pieces, plated on 2% potato dextrose agar, and maintained at 20°C with 14 h of light per day. Colonies with white mycelium developed after 3 days. Microconidia and three-septated macroconidia averaged 11 × 3.9 µm and 38 × 5 µm, respectively. Chlamydospores were produced singly and in pairs. On the basis of culture characteristics, the fungus was identified as Fusarium solani (2). The ITS1-5.8S-ITS2 DNA sequences of 10 monoconidial cultures shared 99% identity with F. solani strains available on the NCBI databases (GenBank Accession Nos. 1115947 and 1115999). Pathogenicity tests were conducted with F. solani isolates NR1 and NR2 obtained from symptomatic plants. Twelve-month-old rooted cuttings of O. europaea cv. Leccino were transferred to pots containing a soilless mix and F. solani-infected oat grains (10:1 vol/vol). Fifteen plants of each F. solani isolate were inoculated. Noninfested sterilized oat grains were used for the control treatment. Symptoms on inoculated plants included leaf abscission followed by wilting and plant death approximately 10 days after inoculation and resembled those observed on the naturally infected plants. Noninoculated control plants remained healthy. The fungus was reisolated from roots of symptomatic tissues and was identical in appearance to the isolates used to inoculate the plants. No colonies of F. solani were isolated from noninoculated plants. F. solani has been reported as weakly pathogenic on olive in Spain (4) and highly aggressive on olive in Argentina (1) and India (3). To our knowledge, this is the first report of F. solani causing wilt and mortality of young olive plants in Nepal. References: (1) S. Babbit et al. Plant Dis. 86:326, 2002. (2) C. Booth. Fusarium Laboratory Guide to the Identification of the Major Species. CMI, Kew, England, 1977. (3) R. L. Munjal et al. Studies on diseases of olive in Himachal Pradesh. Page 437 in: Improvement of Forest Biomass. Symposium Proceedings. Indian Society of Tree Scientists. P. K. Kosla, ed. Sdan, India, 1982. (4) M. E. Sánchez Hernández et al. Eur. J. Plant Pathol. 104:347, 1998.

First Report of Root Rot and Twigs Wilting of Olive Trees in Argentina Caused by Phytophthora nicotianae.

In Argentina, olives (Olea europaea) are planted on approximately 90,000 ha located primarily in the northwest continental regions. During a 2005 survey, root rot was recorded at several olive plantations in Catamarca, La Rioja, and San Juan provinces (3). Aboveground symptoms associated with root rot were twigs wilting with or without chlorosis, defoliation, and death. Symptoms were initiated on lateral branches and sometimes affected the entire crown. Even if young (5-year-old) trees displayed root rot, aerial symptoms may or may not be seen until years later. Disease incidence varied from 3 to 30%. Rotted rootlets were associated mainly with the infection of Phytophthora palmivora Butler and less frequently with another Phytophthora species. Isolates of this species were heterothallic, had a fluffy growth on carrot agar, and arachnoid growth on potato dextrose agar. Chlamydospores approximately 36 µm in diameter were also produced. The species developed prominent, papillate, noncaducous sporangia of different shapes ranging from ellipsoid to spherical when submerged in saline solution. Sporangia were 35 to 57 × 25 to 45 µm (average 44 × 33 µm), L:B ratio from 1.1 to 1.7. Isolates formed oogonia and amphyginous antheridia following mating type assays. On the basis of morphological features, these isolates were identified as P. nicotianae Breda de Haan. Identity was confirmed by sequencing the rDNA internal transcribed spacer (GenBank Accession No. FJ746693) (1). One-year-old O. europea seedlings were challenged with P. nicotianae (A1 isolates 306G and 339) through soil infestation assay in a growth chamber at 25°C. Infested and uninfested autoclaved millet grains moistened with V8 juice were used to inoculate 15 olive seedlings per isolate and controls, respectively. Fifty days after inoculation, seedlings showed foliar symptoms similar to those observed in the field and had an average of 50% reduction in the root system. Control plants remained healthy. P. nicotianae was always reisolated from symptomatic roots. P. nicotianae was reported on Citrus aurantium in Argentina in 1947 and is currently associated with several hosts (2). In 2002, the same species was reported associated with olive root rot in southern Italy (4). It is possible that P. nicotianae was recently introduced into Argentina through importation of Mediterranean olive varieties. The demonstrated pathogenicity of P. nicotianae on olive together with the recently reported detection of P. palmivora (3) presents a serious threat to olive cultivation in Argentina. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) M. J. Frezzi. Rev. Investig. Agric. 4:47, 1950. (3) G. Lucero et al. Plant Pathol. 56:728, 2007. (4) F. Nigro and A. Ippolito. Acta Hortic. 586:777, 2002.

First Report of Phytophthora cactorum Associated With Beech Decline in Italy.

During 2006 and 2007, declining mature beech trees (Fagus sylvatica) were recorded in two stands in the Natural Park of Monti Cimini in central Italy. Symptoms included crown thinning and the presence of bleeding lesions on the main roots and lower stem. Incidence of decline was approximately 5%. Samples of necrotic bark tissue were collected, cut into 5 mm long segments, plated on PARPNH, and incubated at 20°C (1). Phytophthora isolates were obtained from necrotic tissues of 25% of the sampled declining trees. Colonies were rosaceous on potato dextrose agar (PDA) and homothallic. Papillate, ovoid-to-obpyriform, caducous sporangia (mean 38 × 26.2 µm) were produced in soil extract. Oospores were plerotic (mean diameter of 22 to 32 µm) and antheridia paragynous. Optimum growth temperature was 23 to 25°C, minimum 6 to 8°C and maximum 30 to 33°C. A portion of the internal transcribed spacer sequence has been deposited in the NCBI database (GenBank Accession No. FJ183724). A BLAST search of the NCBI database revealed Phytophthora cactorum, Accession No. EU194384, as the closest match with 100% sequence similarity. Pathogenicity of two isolates, PFE3 and IFB-CAC 38, collected from distressed beech trees was tested using a soil infestation test (10 beech seedlings per isolate and control) and an under the bark inoculation method (10 twigs per isolate and controls, wounded and noninoculated taken from a declining beech tree) (2). After 2 weeks at 20°C, twigs and seedlings inoculated with each isolate developed extensive necrotic lesions around the inoculation sites and the collar, respectively, and P. cactorum could be reisolated from all lesions. Controls showed no symptoms. P. cactorum is widespread in declining beech forests in central Europe (1). In Italy, P. cactorum occurs in soils of chestnut and oak forests and was isolated from collar and root lesions of declining walnut trees (3). To our knowledge, this is the first report of P. cactorum being associated with declining beech trees in Italy. References: (1) T. Jung. Forest Pathol. Online publication. doi:10.1111/j.1439-0329.2008.00566.x, 2008. (2) T. Jung et al. Eur. J. For. Pathol. 26:253, 1996. (3) A. M. Vettraino et al. Plant Pathol. 52:491, 2003.

First Report of Root and Collar Rot by Phytophthora cryptogea on Sweet Cherry in Italy.

Sweet cherry (Prunus avium L.) cultivation has increased in Italy and ≈30,000 ha are now in production. In the spring of 2006, a disease survey was conducted in orchards of central Italy. Decline symptoms of Prunus avium 'Durone' grafted on wild rootstocks of Prunus avium consisted of reduced tree vigor, yellowing of leaves, and development of root and collar rot. A reddish brown discoloration of the inner bark and wood was observed on symptomatic trees. Symptoms were recorded in three cherry orchards seasonally submitted to soil saturation conditions. In one orchard, symptoms were observed on approximately 30% of 170 surveyed trees. A Phytophthora species was consistently isolated on PARPH-V8 medium (2) from collar lesions of five cherry trees over nine trees randomly chosen among symptomatic trees in the three orchards. Colonies (20) were isolated and all appeared fluffy on potato dextrose agar and did not grow at 35°C. The morphological characteristics fit the descriptions of Phytophthora cryptogea Pethybr. & Lafferty (1). Sequence analysis of the internal transcribed spacers region of isolates shared 100% homology with P. cryptogea (GenBank Accession Nos. EF418948.1, EF418943.1, and EF153671.1). Pathogenicity of two P. cryptogea isolates (AN1 and AN2, Accession Nos. EF661576 and EF661577, respectively) was tested by soil infestation in a growth chamber at 22°C using 15 1-year-old rooted seedlings of wild rootstock of Prunus avium for each isolate. Noninoculated seedlings (15) were used as negative controls. The inoculum was produced by growing isolates on autoclaved millet grains moistened with V8 juice for 4 weeks. Each plant was inoculated with 30 cm3 of inoculum per liter of soil. Each seedling was randomly assigned to isolates AN1, AN2, or the noninoculated control and arranged in a complete random design. All plants were flooded for 24 h every 2 weeks for 8 weeks. At the end of the experiment, all inoculated seedlings showed crown wilting and root rot. Control plants showed no symptoms. Percentage of necrotic lateral roots expressed as dry weight (±SE) was 19.9 ± 0.6, 17.5 ± 0.2, and 4.4 ± 0.5, respectively, for seedlings inoculated with isolates AN1, AN2, and controls (analysis of variance F = 7.48, P < 0.01). Values of inoculated seedlings were significantly different from the control plants with the Tukey Post-Hoc test. No difference in virulence was observed between the two isolates. P. cryptogea was always reisolated from the roots of inoculated plants. Although pathogenicity of P. cryptogea on sweet cherry has been reported in the United States and other European countries (3,4), to our knowledge, this is the first record of P. cryptogea on sweet cherry orchards in Italy. The presence of P. cryptogea in cherry orchards is of significant concern because of its aggressiveness to this host and woody hosts such as apple, apricot, peach, walnut, and kiwi. References: (1) D. C Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) A. J. Ferguson and S. N. Jeffers. Plant Dis. 83:1129, 1999. (3) T. Tomidis and T. Sotiropoulos. N. Z. J. Crop Hortic. Sci. 31:355, 2003. (4) W. F. Wilcox and S. M. Mircetich. Phytopathology 75:1451, 1985.

A New Report of Phytophthora ramorum on Rhamnus purshiana in Northern California.

Rhamnus purshiana, or cascara, is a deciduous tall shrub or small tree as much as 9 m high with thin, smooth, silver-gray bark. It is often present in shady sites in redwood and mixed evergreen forests of the North Ameri-can west coast, from British Columbia to northern California. In July 2005, symptomatic leaves with irregular, black spots, 2 to 5 mm in diameter and concentrated toward the tips, were collected from four cascara plants in the Samuel P. Taylor State Park, Marin County, California. There was no evidence of defoliation. Pieces of necrotic tissue were plated on selective medium (PARP) and maintained at 19°C for 2 weeks. A Phytophthora sp. was consistently isolated and it was identified as P. ramorum on the basis of morphological and molecular traits published previously (3,4). The P. ramorum isolate Pr-418 has been deposited in the American Type Culture Collection (ATCC MYA-3676) and a portion of the internal transcribed sequence (ITS) of rDNA has been deposited in the NCBI database (GenBank Accession No. DQ168874). Koch's postulates were completed using the leaf-dip method (2) on detached leaves collected from three cascara plants growing at the University of California Botanical Garden at Berkeley. Zoospore inoculum was prepared by flooding a 2-week-old culture growing on V8 agar with sterile water for 4 days. The liquid was filtered after cold shocking at 4°C for 30 min and incubated at room temperature for 1 h. Fifteen leaves were dipped in the resulting zoospore suspension (1.6 × 104 zoospores per ml) for either 1 min (experiment 1) or overnight (experiment 2). Leaves used as negative controls were dipped in sterile water. After removal from the inoculum, excess liquid was allowed to drain. Leaves were maintained in a moist chamber at 19°C with 13 h of natural light for 1 week. After 3 days of incubation, necrotic spots similar to those observed in the field had developed on leaves in experiment 2, while no symptoms were observed in experiment 1. Necrotic lesions were observed on 12 and 15 of 15 leaves in experiments 1 and 2, respectively, after 7 days of incubation. For each leaf, the necrotic area and percent necrosis was determined by placing the leaves in a flatbed scanner and processing the images with Assess (Version 1.01; The American Phytopathological Society, St. Paul, MN). Lesions extended from the tip of the leaves and covered 3 ± 1% of the total leaf area in experiment 1 and 33 ± 3% in experiment 2. Reisolation of P. ramorum on PARP was successful for all inoculated leaves. P. ramorum was never isolated from negative controls and no symptoms of infection were observed. The leaf-dip inoculation method is a rapid and reliable indicator of host susceptibility to P. ramorum, with many species developing necrosis when exposed to high concentrations of zoospores (3). Our results show that exposure time to the pathogen can play an important role in the development of symptoms. R. purshiana has been previously reported as a host in Oregon (1,2), but to our knowledge, this is the first report of cascara as a natural host of P. ramorum in the state of California. Our results confirm those from Oregon (2). The impact of infection by P. ramorum on cascara is unknown. References: (1) J. M. Davidson et al. Plant Health Prog. DOI:10.1094/PHP-2003-0707-01-DG, 2003. (2) E. Hansen et al. Plant Dis. 89:63, 2005. (3) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (4) S. Werres et al. Mycol Res. 105:1155, 2001.

First Report of Infection of Maiden-Hair Fern (Adiantum jordanii and A. aleuticum) by Phytophthora ramorum in California.

During July 2005, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from nine native Adiantum jordanii plants growing at two forest sites (Samuel P. Taylor State Park, Marin County and Peachland Road, Mendocino County) and from seven A. aleuticum plants at one forest site (Peachland Road) in California. At both locations, symptomatic plants were distributed close to rivers and roads and in association with infected bay laurel trees (Umbellularia californica), toyon (Heteromeles arbutifolia), and tanoaks (Lithocarpus densiflorus). Symptomatic leaflets showed brown spots that sometimes coalesced, killing entire leaves, but the disease did not appear to be fatal to the ferns. Necrotic tissues were plated on PARP and maintained in the dark at 18°C for 1 to 2 weeks. Isolates were identified as P. ramorum on the basis of colony morphology, the presence of chlamydospores and caducous, semipapillate sporangia, and the internal transcribed spacer (ITS) rDNA sequences (1,2). The P. ramorum isolates, Pr-419 from A. jordanii and Pr-422 from A. aleuticum, have been deposited in the American Type Culture Collection (ATCC MYA-3677 and MYA-3679, respectively) and a region of the ITS rDNA sequence deposited in the NCBI database (GenBank Accession No. DQ173082 and DQ219821, respectively). To test the pathogenicity, the tips of freshly detached leaves of A. jordanii and A. aleuticum were dipped into a solution of 1 × 103 zoospores per ml of Pr-419 and Pr-422 for 1 min. The wounded end of the leaves was not exposed to the inoculum. The zoospores were produced by flooding agar disks (1 cm in diameter) from the margin of 8- to 14-day-old colonies growing on V8 juice agar with sterile deionized water. After 3 days of incubation at 20°C in the dark, zoospore release was induced by placing dishes at 4°C for 20 min and then at room temperature for 60 min. For each Adiantum species and P. ramorum isolate, 15 leaves collected from five potted nursery plants were tested. Control leaves were dipped in sterile deionized water. Leaves were maintained in a moist chamber at 19°C with 13 h of natural light for 9 days. Brown lesions similar to those seen in the forest developed on approximately 60 and 33% of the A. jordanii and A. aleuticum leaves, respectively, inoculated with Pr-419 and on approximately 73 and 40% of the leaves inoculated with Pr-422. Under these experimental conditions, A. aleuticum appeared to be slightly more susceptible than the A. jordanii, with a necrotic leaf area of approximately 38% compared with 20%. The pathogen was reisolated on PARP after surface sterilization from all symptomatic leaves. Control leaves did not develop symptoms and P. ramorum was not recovered. A. jordanii and A. aleuticum have already been listed as associated hosts for P. ramorum on the APHIS (USDA Animal and Plant Health Inspection Service) website ( http://www.aphis.usda.gov/ ). To our knowledge, this is the first report of ferns as natural hosts of P. ramorum. References: (1) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (2) S. Werres et al. Mycol Res. 105:1155, 2001.

A microbiology investigation on probiotic pharmaceutical products used for human health.

Many and different probiotic pharmaceutical products are presently commercialised in the world. On this regard, a microbiological investigation was carried out to screen the microorganisms incorporated into these products, commonly used for human health. After determination of the cell number and viability of bacteria, several experiments were performed in vitro in order to characterise the microorganisms and to evaluate their probiotic value. Among all the strains identified, best results were obtained with Lactobacillus rhamnosus, Enterococcus faecium and Saccharomyces cerevisiae as far as regards growth rates, pH and bile salts tolerance. Moreover, the identification profiles of microorganisms showed a better reliability for the products containing a single species whereas the ones composed of different strains were usually not satisfactory. In some cases, the presence of Lactobacillus and Saccharomyces species was in disagreement with the claimed composition of the product and some species of lactobacilli, bifidobacteria and streptococci were found not viable. In defined mixed cultures experiments, the antagonism of Lactobacillus acidophilus and Enterococcus faecium versus Yersinia enterocolitica was demonstrated and explained as acid and/or antimicrobials production.

First Report of Phytophthora cryptogea in Walnut Stands in Italy.

English (Persian) walnut (Juglans regia L.) is among the most widely cultivated species in pure and mixed plantations of broadleaved trees in Italy. A decline of walnut of increasing occurrence has been reported recently in new plantations in central and northern Italy. Symptoms of the decline were typically characterized by yellowing of the foliage, defoliation, and plant death. Dark, flame-shaped necroses were often present at the collar. Phytophthora cactorum, P. cambivora, and P. cinnamomi were among the species associated with necrotic tissues of the collar and main roots (1). Furthermore, a Phytophthora sp. was isolated from soil removed from the lateral root zone of 6 of 15 declining trees in 3 walnut plantations, 2 in northern Italy and 1 in central Italy. Isolations were made by baiting with Rhododendron leaves and plating on PARBhy selective medium (3). The species isolated was identified as P. cryptogea on the basis of morphological and cultural characteristics (2). All isolates produced oval to obpyriform, nonpapillate sporangia and were mating type A2. Identification of the isolates was confirmed by comparing the restriction fragment length polymorphism patterns of the internal transcribed spacer region of ribosomal DNA with those obtained from previously identified Phytophthora species. Pathogenicity tests on potted 2-year-old walnut seedlings were conducted using two isolates of P. cryptogea. Inoculum was prepared by growing isolates on sterilized millet seeds added to soil at 2.5% (wt/vol). Sporulation was induced by 24-h flooding of the soil. Symptoms were assessed 1 month after inoculation. Ten uninoculated seedlings were used as controls. Inoculated seedlings showed no symptoms on the tap root, but there were extensive necroses of lateral roots ranging from 14 to 75% (average 38.6 ± 6.7 SE) of total lateral root (dry weight) compared with values of 0 to 11% (average 3 ± 1.5 SE) for uninoculated seedlings. P. cryptogea was easily reisolated from infected lateral roots and from the soil of inoculated pots. The inoculation trials confirmed P. cryptogea as a feeder-root pathogen of walnut in Italy. To our knowledge, this is the first report of P. cryptogea on English walnut in Italy. This species often has been associated with walnut decline in the United States (2) and on other woody plants in Italy (3). References: (1) A. Belisario et al. Petria 11:127, 2001. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Association, St. Paul, MN, 1996. (3) A. M. Vettraino et al. Plant Pathol. 50:90, 2001.

First Report of Phytophthora nicotianae and P. citricola Associated with English Walnut Decline in Europe.

English (Persian) walnut (Juglans regia), among the most widely cultivated species of Juglans worldwide, is cultivated primarily for fruit production but also for timber. In the last 10 years, walnut decline causing leaf yellowing, sparse foliage, overall decline, and plant death has increased in Italian commercial orchards. In Italy, Phytophthora cactorum, P. cambivora, P. cinnamomi, and P. cryptogea are associated with this disease (1,4). Over the last 5 years, P. cinnamomi was the most widely isolated and destructive species (1). Recently, a different species of Phytophthora was isolated from diseased roots and soil from around lateral roots of 10 declining trees in two orchards in the Veneto Region of northern Italy. Another species of Phytophthora was isolated consistently from rotted roots of declining walnut trees in two orchards in the Campania Region of southern Italy. Phytophthora spp. were isolated directly from plant material or Rhododendron spp. leaf baiting on soil samples with PARBhy selective medium (10 mg of pimaricin, 250 mg of ampicillin [sodium salt], 10 mg of rifampicin, 50 mg of hymexazol, 15 mg of benomyl, 15 g of malt extract, 20 g of agar in 1,000 ml of H2O). Two species of Phytophthora were identified based on morphological and cultural characteristics (2). The species from trees in the Veneto Region was identified as P. nicotianae. All isolates produced papillate, spherical to obturbinate, occasionally caducous sporangia with short pedicels, terminal and intercalary chlamydospores, and were mating type A2. The species isolated from trees in the Campania Region was identified as P.citricola. Isolates were homothallic, produced semipapillate, persistent, obclavate to obpyriform sporangia, occasionally with two apices, and antheridia paragynous. Identifications were confirmed by comparing restriction fragment length polymorphism patterns of the internal transcribed spacer region of rDNA with those obtained from previously identified species of Phytophthora. Pathogenicity of two isolates each of P. citricola and P. nicotianae was tested on 2-year-old potted walnut seedlings. Inocula were prepared by inoculating sterilized millet seeds moistened with V8 broth with plugs of mycelium and incubated for 4 weeks at 20°C in the dark. Infested seeds were added to potting soil at a rate of 3% (wt/vol). One day later, pots were flooded for 48 h to promote sporulation. Ten noninoculated seedlings were used as the control. Symptoms were assessed 2 months after inoculation. Seedlings inoculated with P. nicotianae developed necrosis of feeder and lateral roots, but only limited infection of taproots. Seedlings inoculated with P. citricola developed necroses at the insertion points of lateral roots. All four isolates produced visible damage to lateral roots on inoculated plants. P. nicotianae and P. citricola were reisolated from respectively infected roots. Results from these inoculations confirmed P. nicotianae and P. citricola as root pathogens of English walnut. Both species were associated with walnut decline as reported in the United States (3). To our knowledge, this is the first report of P. nicotianae and P. citricola on J. regia in Europe. References: (1) A. Belisario et al. Petria 11:149. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) M. E. Matheron and S. M. Mircetich. Phytopathology 75:977, 1985. (4) A. M. Vettraino et al. Plant Dis. 86:328, 2002.

Early detection of Biscogniauxia nummularia in symptomless European beech (Fagus sylvatica L.) by TaqMan quantitative real-time PCR.

AIMS: To develop a quantitative real-time PCR (Rt PCR) assay for the early detection of Biscogniauxia nummularia, a xylariaceous fungus that causes strip-canker and wood decay on European beech (Fagus sylvatica L.). METHODS AND RESULTS: The molecular assay was based on TaqMan chemistry using species-specific primers and a fluorogenic probe designed on the ITS1 sequence of rRNA gene clusters. The specificity of the oligonucleotides and the probe were tested using the DNA of B. nummularia isolates from different geographic areas, of phylogenetically related species, and of some fungi commonly colonizing European beech bark and wood. A total of 31 symptomless and symptomatic shoots of European beech were collected from three forest sites in the Apennine Mountains of Italy. The percentage of positive detections of B. nummularia with the TaqMan assay was 78.6%, compared with only 14.3% of positive isolations on growth media for two sites. CONCLUSIONS: In shoots, the quantitative Rt PCR assay detected down to 8.0-fg fungal DNA per microgram of total DNA extracted. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed in quantitative Rt PCR, by using TaqMan chemistry, revealed a rapid and sensitive method useful for the early detection of B. nummularia in symptomless European beech twigs.