Effects of Soil Solarization and Trichoderma asperellum on Soilborne Inoculum of Phytophthora ramorum and Phytophthora pini in Container Nurseries.

Infested container nursery beds are an important source of soilborne Phytophthora spp. for initiating disease through movement with surface water or splashing onto foliage. We investigated the effects of soil solarization, alone or with subsequent amendment with a Trichoderma asperellum biocontrol agent, on the survival of Phytophthora spp. inoculum. In field trials conducted with Phytophthora ramorum in San Rafael, CA and with P. pini in Corvallis, OR, infested rhododendron leaf inoculum was buried at 5, 15, and 30 cm below the soil surface. Solarization for 2 or 4 weeks during summer 2012 eliminated recovery of Phytophthora spp. buried at all depths in California trial 1, at 5 and 15 cm in California trial 2, but only at 5 cm in Oregon. There was no significant reduction of Phytophthora spp. recovery after T. asperellum application. Although the population densities of the introduced T. asperellum at the 5-cm depth were often two- to fourfold higher in solarized compared with nonsolarized plots, they were not significantly different (P = 0.052). Soil solarization appears to be a promising technique for disinfesting the upper layer of soil in container nurseries under certain conditions.

Phytophthora ramorum Colonizes Tanoak Xylem and Is Associated with Reduced Stem Water Transport.

ABSTRACT Isolation, detection with diagnostic polymerase chain reaction (PCR), and microscopy demonstrated the presence of Phytophthora ramorum in the sapwood of mature, naturally infected tanoak (Lithocarpus densiflorus) trees. The pathogen was strongly associated with discolored sapwood (P < 0.001), and was recovered or detected from 83% of discolored sapwood tissue samples. Hyphae were abundant in the xylem vessels, ray parenchyma, and fiber tracheids. Chlamydospores were observed in the vessels. Studies of log inoculation indicated that P. ramorum readily colonized sapwood from inoculum placed in the bark, cambium, or sapwood. After 8 weeks, radial spread of P. ramorum in sapwood averaged 3.0 to 3.3 cm and axial spread averaged 12.4 to 18.8 cm. A field study was conducted to determine if trees with infected xylem had reduced sap flux and reduced specific conductivity relative to noninfected control trees. Sap flux was monitored with heat-diffusion sensors and tissue samples near the sensors were subsequently tested for P. ramorum. Adjacent wood sections were excised and specific conductivity measured. Both midday sap flux and specific conductivity were significantly reduced in infected trees versus noninfected control trees. Vessel diameter distributions did not differ significantly among the two treatments, but tyloses were more abundant in infected than in noninfected trees. Implications for pathogenesis, symptomology, and epidemiology are discussed.

Root and Stem Infection of Rhododendron from Potting Medium Infested with Phytophthora ramorum.

Phytophthora ramorum has been detected in soil and potting media, but the potential for root infections is not fully understood. To determine whether the root system could become infected and transmit disease, rhododendron 'Nova Zembla' plants grown from rooted cuttings and native Pacific rhododendron (Rhododendron macrophyllum) plants grown from seed were transplanted into a potting medium artificially infested with P. ramorum. Inoculum consisted of V8-brothvermiculite cultures of P. ramorum, chopped infected leaves, or zoospores. Plants were watered from the bottom to prevent splash dispersal of inoculum onto stems and foliage. Both infested amendments and applications of zoospores resulted in plant mortality within 3 to 7 weeks. P. ramorum was isolated from hair roots, large roots, and stems above and below the potting medium surface. Noninoculated control plants remained healthy and did not yield P. ramorum. Epifluorescence microscopy of tissue culture plantlets inoculated in vitro revealed attraction of zoospores to wounds and root primordia, and colonization of the cortex and vascular tissues of roots and stems, including the xylem. Transmission of P. ramorum from infested potting media to stems via infected, symptomless root tissue demonstrates the need to monitor potting media for presence of the pathogen to prevent spread of P. ramorum on nursery stock.

Susceptibility of Oregon Forest Trees and Shrubs to Phytophthora ramorum: A Comparison of Artificial Inoculation and Natural Infection.

Phytophthora ramorum is an invasive pathogen in some mixed-hardwood forests in California and southwestern Oregon, where it causes sudden oak death (SOD) on some members of Fagaceae, ramorum shoot dieback on some members of Ericaceae and conifers, and ramorum leaf blight on diverse hosts. We compared symptoms of P. ramorum infection resulting from four different artificial inoculation techniques with the symptoms of natural infection on 49 western forest trees and shrubs; 80% proved susceptible to one degree or another. No single inoculation method predicted the full range of symptoms observed in the field, but whole plant dip came closest. Detached-leaf-dip inoculation provided a rapid assay and permitted a reasonable assessment of susceptibility to leaf blight. Both leaf age and inoculum dose affected detached-leaf assays. SOD and dieback hosts often developed limited leaf symptoms, although the pattern of midrib and petiole necrosis was distinctive. Stem-wound inoculation of seedlings correlated with field symptoms for several hosts. The results suggested that additional conifer species may be damaged in the field. Log inoculation provided a realistic test of susceptibility to SOD, but was cumbersome and subject to seasonal variability. Pacific rhododendron, salmonberry, cascara, and poison oak were confirmed as hosts by completing Koch's postulates. Douglas-fir was most susceptible to shoot dieback shortly after budburst, with infection occurring at the bud.

Detection of Phytophthora ramorum Blight in Oregon Nurseries and Completion of Koch's Postulates on Pieris, Rhododendron, Viburnum, and Camellia.

Phytophthora ramorum, the cause of sudden oak death in California and Oregon coastal forests and ramorum blight in European nurseries and landscapes (1), was detected in six Oregon nurseries in Jackson, Clackamas, and Washington counties from May to June 2003. The pathogen was isolated from: Viburnum bodnantense 'Dawn', V. plicatum var. tomentosum 'Mariesii', Pieris japonica × formosa 'Forest Flame', P. japonica 'Variegata' and 'Flaming Silver', P. floribunda × japonica 'Brouwer's Beauty', Camellia sasanqua 'Bonanza' and other cultivars, C. japonica, and Rhododendron × 'Unique'. Samples of symptomatic tissues were plated on a Phytophthora-selective medium (PARP) and tested by polymerase chain reaction (PCR) (3). All samples positive for P. ramorum with PCR yielded P. ramorum isolates in culture. The isolates have the European genotype, mating type A1, except for the Camellia spp. isolates, which have the North American genotype, mating type A2 (2). Isolates are deposited in the American Type Culture Collection. Koch's postulates for this pathogen have been completed on V. bodnantense and C. japonica (1). To confirm pathogenicity on the new hosts, isolates from V. plicatum var. tomentosum 'Mariesii', Pieris × 'Forest Flame', Pieris × 'Brouwer's Beauty', and P. japonica 'Variegata' and 'Flaming Silver' were used to inoculate healthy plants of the same cultivars. For isolates from Rhododendron × 'Unique' and C. sasanqua 'Bonanza', pathogenicity was tested on Rhododendron × 'Nova Zembla' and C. sasanqua 'Sutsugekka' and 'Kanjiro'. Three to five plants of each cultivar were inoculated and three to five were noninoculated. Zoospore inoculum was prepared on dilute V8 agar for one isolate from each host. Foliage of plants growing in 10-cm pots was dipped for 5 sec in a zoospore suspension (3 × 104 zoospores per ml) or sprayed to runoff with a hand mister (6 × 104 zoospores per ml). Control plants were dipped in or sprayed with sterile water. C. sasanqua plants were also inoculated by placing 6-mm mycelial plugs on individual leaves that had been wounded by piercing with a pin. Control leaves were wounded but not inoculated. Foliage was enclosed in plastic bags to retain humidity and the pathogen, and plants were incubated in a locked growth chamber (21 to 23°C). After 21 days, plants were examined for symptoms, and isolations onto PARP were made. All inoculated plants showed foliar symptoms, and P. ramorum was consistently isolated from inoculated plants, but not from asymptomatic control plants. On Rhododendron × 'Nova Zembla', nearly all leaves were wilted and dead, as were terminal buds and stems. Pieris spp. cultivars exhibited leaf and stem necrosis and defoliation. On V. plicatum var. tomentosum 'Mariesii', necrotic leaf lesions and defoliation of the lower leaves were observed. On C. sasanqua, necrotic lesions developed only on wounded leaves inoculated with mycelial plugs; these leaves abscised. Our results confirm the pathogenicity of Oregon nursery isolates of P. ramorum on V. plicatum var. tomentosum 'Mariesii', P. japonica × formosa 'Forest Flame', P. japonica 'Variegata' and 'Flaming Silver', P. floribunda × japonica 'Brouwer's Beauty', C. sasanqua and Rhododendron and complete Koch's postulates for several new hosts. References: (1) J. M. Davidson et al. Online publication. doi:10.1094/PHP-2003-0707-01-DG. Plant Health Progress, 2003. (2) E. M. Hansen et al. Plant Dis. 87:1267, 2003. (3) L. M. Winton and E. M. Hansen. For. Pathol. 31:275, 2001.

Diversity of the Burkholderia cepacia complex and implications for risk assessment of biological control strains.

The Burkholderia cepacia complex (Bcc) consists of several species of closely related and extremely versatile gram-negative bacteria found naturally in soil, water, and the rhizosphere of plants. Strains of Bcc have been used in biological control of plant diseases and bioremediation, while some strains are plant pathogens or opportunistic pathogens of humans with cystic fibrosis. The ecological versatility of these bacteria is likely due to their unusually large genomes, which are often comprised of several (typically two or three) large replicons, as well as their ability to use a large array of compounds as sole carbon sources. The original species B. cepacia has been split into eight genetic species (genomovars), including five named species, but taxonomic distinctions have not enabled biological control strains to be clearly distinguished from human pathogenic strains. This has led to a reassessment of the risk of several strains registered by the U.S. Environmental Protection Agency for biological control. We review the biology of Bcc bacteria, especially how our growing knowledge of Bcc ecology and pathogenicity might be used in risk assessment. The capability of this bacterial complex to cause disease in plants and humans, as well as to control plant diseases, affords a rare opportunity to explore traits that may function in all three environments.

Postinfection Biological Control of Oomycete Pathogens of Pea by Burkholderia cepacia AMMDR1.

ABSTRACT Burkholderia cepacia AMMDR1 is a biocontrol agent that reduces Pythium damping-off and Aphanomyces root rot severity on peas in the field. We studied the effect of B. cepacia AMMDR1 on post-infection stages in the life cycles of these pathogens, including mycelial colonization of the host, production of oogonia, and production of secondary zoospore inoculum. We used Burkholderia cepacia 1324, a seed and rootcolonizing but antibiosis-deficient Tn5 mutant of B. cepacia AMMDR1, to study mechanisms of biological control other than antibiosis. B. cepacia AMMDR1 significantly reduced Pythium aphanidermatum postinfection colonization and damping-off of pea seeds, even when the bacteria were applied 12 h after zoospore inoculation. B. cepacia AMMDR1 also significantly reduced colonization of taproots by Aphanomyces euteiches mycelium, but only when the bacteria were applied at high population densities at the site of zoospore inoculation. The antibiosisdeficient mutant, B. cepacia 1324, had no effect on mycelial colonization of seeds or roots by Pythium aphanidermatum nor A. euteiches, suggesting that antibiosis is the primary mechanism of biological control. B. cepacia AMMDR1, but not B. cepacia 1324, reduced production of A. euteiches oogonia. This effect occurred even when the population size of B. cepacia AMMDR1 was too small to cause a reduction in lesion length early on in the infection process and may result from in situ antibiotic production. B. cepacia AMMDR1 had no effect on the production of secondary zoospores of A. euteiches from infected roots. The main effects of B. cepacia AMMDR1 on postinfection stages in the life cycles of these pathogens therefore were reductions in mycelial colonization by Pythium aphanidermatum and in formation of oogonia by A. euteiches. No mechanism other than antibiosis could be identified.

Zoospore homing and infection events: effects of the biocontrol bacterium Burkholderia cepacia AMMDR1 on two oomycete pathogens of pea (Pisum sativum L.).

Burkholderia cepacia AMMDR1 is a biocontrol agent that protects pea and sweet corn seeds from Pythium damping-off in field experiments. The goal of this work was to understand the effect of B. cepacia AMMDR1 on Pythium aphanidermatum and Aphanomyces euteiches zoospore homing events and on infection of pea seeds or roots. In vitro, B. cepacia AMMDR1 caused zoospore lysis, prevented cyst germination, and inhibited germ tube growth of both oomycetes. B. cepacia AMMDR1 also reduced the attractiveness of seed exudates to Pythium zoospores to nondetectable levels. However, when present at high levels on seeds, B. cepacia AMMDR1 had little net effect on zoospore attraction, probably because it also enhanced seed exudation. Seed-applied B. cepacia AMMDR1 dramatically reduced the incidence of infection by Pythium zoospores in situ compared with an antibiosis-deficient Tn5 mutant strain. This mutant strain also decreased Pythium infection incidence to some extent, but only when the pathogen inoculum potential was low. B. cepacia AMMDR1 did not affect attraction of Aphanomyces zoospores or Aphanomyces root rot incidence. These results suggest that B. cepacia AMMDR1 controls P. aphanidermatum largely through antibiosis, but competition for zoospore-attracting compounds can contribute to the effect. Differences in suppression of Aphanomyces and Pythium are discussed in relation to differences in the ecology of the two pathogens.