Risk of Epidemic Development in Nurseries from Soil Inoculum of Phytophthora ramorum.

Soilborne inoculum arising from buried, infested leaf debris may contribute to the persistence of Phytophthora ramorum at recurrently positive nurseries. To initiate new epidemics, inoculum must not only survive but also produce sporangia during times conducive to infection at the soil surface. To assess this risk, we performed two year-long experiments in a soil plot at the National Ornamentals Research Site at Dominican University of California. Inoculated rhododendron leaf disks were buried at a depth of 5 or 15 cm in the early summer of 2014 or 2015. Inoculum was baited at the soil surface with noninfested leaf disks (2014 only) and then retrieved to assess pathogen viability and sporulation capacity every 5 weeks. Two 14-week-long trials were conducted in 2016. We were able to consistently culture P. ramorum over all time periods. Soil incubation rapidly reduced the capacity of inoculum to sporulate, especially at 5 cm; however, sporulation capacity increased with the onset of seasonally cooler temperatures. P. ramorum was baited most frequently between November and January, especially from inoculum buried at 5 cm 1 day before the baiting period; in January we also baited P. ramorum from inoculum buried at 15 cm the previous June. We validate prior observations that P. ramorum poses a greater risk after exposure to cooler temperatures and provide evidence that infested leaf debris plays a role in the perpetuation of P. ramorum in nurseries. This work provides novel insights into the survival and epidemic behavior of P. ramorum in nursery soils.

Root Rot of Juniperus and Microbiota by Phytophthora lateralis in Oregon Horticultural Nurseries.

Widespread symptoms of root rot and mortality on Juniperus communis and Microbiota decussata were observed in two horticultural nurseries in Oregon, leading to the isolation of a Phytophthora sp. from diseased roots. Based on morphology and sequencing the internal transcribed spacer ITS1-5.8S-ITS2 region, isolates were identified as the invasive pathogen Phytophthora lateralis, causal agent of Port-Orford-cedar (POC; Chamaecyparis lawsoniana) root disease. Additional sequencing of the cytochrome c oxidase subunit 1 and 2 genes identified all isolates as belonging to the PNW lineage. Utilizing recovered isolates plus a POC-wildlands isolate and susceptible POC as controls, we completed Koch's postulates on potted Juniperus and Microbiota plants. Nursery isolates were more aggressive than the forest isolate, which was used in the POC resistance breeding program. Increased aggressiveness was confirmed using a branch stem dip assay with four POC clones that differed in resistance, although no isolate completely overcame major-gene resistance. Isolates were sensitive to mefenoxam, a fungicide commonly used to suppress Phytophthora spp. growth in commercial nurseries. Although POC resistance is durable against these more aggressive nursery isolates, the expanded host range of P. lateralis challenges POC conservation through the continued movement of P. lateralis by the nursery industry.

Film-Forming Polymers and Surfactants Reduce Infection and Sporulation of Phytophthora ramorum on Rhododendron.

Phytophthora ramorum, cause of sudden oak death and ramorum leaf blight, can persist undetected in infested nurseries. Many conventional fungicides are effective in reducing or delaying symptom expression but some may confound visual detection of infected plants. We tested film-forming polymers (FFPs) and surfactants for their ability to reduce infection and sporulation of P. ramorum on rhododendron. FFPs (Anti-Stress, Moisturin, Nature Shield, Nu-Film, and Vapor Gard) and surfactants (Tergitol, Zonix, and an unregistered AGAE product) were screened in detached-leaf assays. Anti-Stress, Nu-Film, Zonix, and a Nu-Film-Zonix mixture were additionally tested for durability, protection against exposure to infested water, and a reduction in sporulation. FFP effectiveness was retained for at least 3 weeks of exposure to overhead irrigation and rain. Relative to controls, foliar treatments protected rhododendron branches exposed to infested water. No treatments prevented symptom development when applied postinfection but leaves treated with Anti-Stress, Zonix, and the Nu-Film-Zonix mixture produced significantly fewer sporangia relative to controls. Application of FFPs and surfactants to quarantined, potentially infected plants offers a management tool for reducing infection and sporulation but not symptom expression, thereby limiting disease spread without interfering with disease detection.

Thermal Inactivation of Inoculum of Two Phytophthora Species by Intermittent Versus Constant Heat.

Research on solarization efficacy has examined the critical temperature and minimum exposure time to inactivate soilborne pathogens. Most mathematical models focus on survival of inoculum subjected to a constant heat regime rather than an intermittent heat regime that better simulates field conditions. To develop a more accurate predictive model, we conducted controlled lab experiments with rhododendron leaf disks infested with Phytophthora ramorum and P. pini. Focused in vitro experiments with P. ramorum showed significantly longer survival of inoculum exposed to intermittent versus constant heat, indicating that intermittent heat is less damaging. A similar trend was observed in soil. Damage was evaluated by comparing the reduction in subsequent survival time of inoculum subjected to different intensities of sublethal heat treatments. Inoculum exposure to continuous heat reflected an increasing rate of damage accumulation. Multiple sublethal heat events resulted in a constant rate of damage accumulation which allowed us to calculate total damage as the sum of damage from each heat event. A model including a correction for an intermittent heat regime significantly improved the prediction of thermal inactivation under a temperature regime that simulated field conditions.

Phytophthora community structure analyses in Oregon nurseries inform systems approaches to disease management.

Nursery plants are important vectors for plant pathogens. Understanding what pathogens occur in nurseries in different production stages can be useful to the development of integrated systems approaches. Four horticultural nurseries in Oregon were sampled every 2 months for 4 years to determine the identity and community structure of Phytophthora spp. associated with different sources and stages in the nursery production cycle. Plants, potting media, used containers, water, greenhouse soil, and container yard substrates were systematically sampled from propagation to the field. From 674 Phytophthora isolates recovered, 28 different species or taxa were identified. The most commonly isolated species from plants were Phytophthora plurivora (33%), P. cinnamomi (26%), P. syringae (19%), and P. citrophthora (11%). From soil and gravel substrates, P. plurivora accounted for 25% of the isolates, with P. taxon Pgchlamydo, P. cryptogea, and P. cinnamomi accounting for 18, 17, and 15%, respectively. Five species (P. plurivora, P. syringae, P. taxon Pgchlamydo, P. gonapodyides, and P. cryptogea) were found in all nurseries. The greatest diversity of taxa occurred in irrigation water reservoirs (20 taxa), with the majority of isolates belonging to internal transcribed spacer clade 6, typically including aquatic opportunists. Nurseries differed in composition of Phytophthora communities across years, seasons, and source within the nursery. These findings suggest likely contamination hazards and target critical control points for management of Phytophthora disease using a systems approach.

A Systems Approach for Management of Pests and Pathogens of Nursery Crops.

Horticultural nurseries are heterogeneous and spatially complex agricultural systems, which present formidable challenges to management of diseases and pests. Moreover, nursery plants shipped interstate and internationally can serve as important vectors for pathogens and pests that threaten both agriculture and forestry. Current regulatory strategies to prevent this movement of pathogens and pests with nursery plants are based on visual inspections of plants just before shipping, a process that is costly and inadequate. Here we propose the application of a systems approach for horticultural nurseries modeled after the Hazard Analysis of Critical Control Points (HACCP) approach widely used in the food processing industry. We evaluated aspects of the systems approach to analyze contamination hazards by Phytophthora species and then implemented management practices targeting specific critical control points. The systems approach for analyzing and correcting unsafe practices offers a potential alternative strategy for preventing plant contamination that could be broadly applied to many pests and pathogens. One of the hallmarks of this approach is its flexibility, including the ability to improve and adapt the approaches as new technologies are developed or improved and to provide the grower with management options.

Phytophthora-ID.org: A Sequence-Based Phytophthora Identification Tool.

Contemporary species identification relies strongly on sequence-based identification, yet resources for identification of many fungal and oomycete pathogens are rare. We developed two web-based, searchable databases for rapid identification of Phytophthora spp. based on sequencing of the internal transcribed spacer (ITS) or the cytochrome oxidase (cox) 1 and 2 spacer region, followed by BLAST searching the databases. Both databases are highly selective. For ITS, only sequences associated with published Phytophthora spp. descriptions or classic Phytophthora phylogenetics references are included. For the cox spacer region, only data obtained by resequencing select isolates reported in published work were included. Novel taxa tentatively named are selectively included in the database and labeled as Phytophthora taxon "X"; as in, for example, P. taxon "asparagi". The database was validated with 700 Phytophthora isolates collected from nursery environments during 2006 to 2009. This resource, found at www.Phytophthora-ID.org , is a robust and validated tool for molecular identification of Phytophthora spp. and is regularly being updated.

The population of oomycetes in a recycled irrigation water system at a horticultural nursery in southern California.

Recapture and recycling of irrigation water is often required to meet enormous water demands at horticultural nurseries. We tested four water types associated with a recycled irrigation system at a commercial container nursery in southern California for presence of oomycete plant pathogens from July 2015 to December 2017. These water types included: the main source of water originating from a reservoir, retention water from an on-site collection pond, irrigation water received by different growing areas within the nursery, and irrigation runoff captured in polyethylene sheet-lined runoff channels. The genera Phytophthora, Pythium, and Phytopythium together contributed more than 85% of the total oomycete population detected in the recycled irrigation system. The Phytophthora and Pythium genera were represented by member species from nine (1-4, 6-10) and eight (A, B, D-F, H-J) different sub-generic clades, respectively. Incoming water sourced from the reservoir was found to harbor known plant pathogens such as Phytophthora citricola-complex, P. capsici-cluster, P. tropicalis,P citrophthora-cluster, P. nemorosa-cluster, P. riparia, P. cryptogea-complex, P. parsiana-cluster, P. sp. nov. aff. kernoviae, Pythium dissotocum-complex, Py. oligandrum-cluster, Py. irregulare, and Phytopythium litorale. Runoff water showed the highest oomycete species richness and frequency of detection with both filtration and leaf baiting methods. In addition to plant pathogens, oomycete fish pathogens such as Aphanomyces laevis, Pythium chondricola-complex, Pythium flevoense-complex, and Saprolegnia diclina-complex were also detected in greater abundance in the recycled irrigation water. The oomycete species richness in the runoff water was correlated with several environmental parameters such as soil temperature. Greater oomycete richness in incoming water was associated with higher soil temperatures, whereas richness in runoff declines with increasing soil temperature, likely suggesting connections to weather-dependent nursery operations.

Standardizing the nomenclature for clonal lineages of the sudden oak death pathogen, Phytophthora ramorum.

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.

Pattern swimming of Phytophthora citricola zoospores: an example of microbial bioconvection.

The genus Phytophthora, belonging to the class Oomycota, comprises a group of over fifty fungus-like plant pathogens in both managed and unmanaged ecosystems. A unique feature of the oomycete lifecycle is a zoosporic stage in which motile, unicellular propagules, serving as the primary agents of dispersal, are produced and released in the presence of water. In Petri dish suspensions, zoospores frequently exhibit 'pattern swimming', whereby they spontaneously form concentrated swimming masses, visible to the naked eye, even in the absence of a chemical or electrical gradient. The nature of this behaviour is unclear, but is of interest because of the potential for auto-attraction and implications for cohort recruitment during infection. Similar behaviour observed in a variety of motile bacteria, algae, and protists is attributed to 'bioconvection' that results from instability in fluid density due to the organisms' upward-swimming tendency and greater-than-water density. In this investigation, we determined that Phytophthora citricola zoospore 'pattern swimming' is unrelated to phototaxis, surface tension-driven (Marangoni) convection, or auto-attraction and that the observed convective pattern, directional swimming, and depth- and concentration dependence are consistent with bioconvection.

Culture-based and non-growth-dependent detection of the Burkholderia cepacia complex in soil environments.

Burkholderia cepacia complex (Bcc) bacteria reside in soil, plant rhizospheres, and water, but their prevalence and distribution in outdoor environments is not clear. We sampled a variety of soil and rhizosphere environments with which people may have contact: playgrounds, athletic fields, parks, hiking trails, residential yards, and gardens. A total of 91 sites was sampled in three large U.S. cities. In the first phase of the study, putative Bcc isolates were recovered on Burkholderia cepacia selective agar and trypan blue tetracycline medium and subsequently examined for biochemical reactivity and growth at 32 and 22 degrees C. Isolates were further examined by PCR assays targeting Bcc-specific ribosomal DNA and recA gene sequences. Among the 1,013 bacterial isolates examined, 68 were identified as Bcc; 14 (15%) of 91 sampled sites yielded Bcc isolates. In the second phase, DNA was extracted directly from soil samples and examined with PCR assays targeting Bcc 16S rRNA gene sequences. Either 82 or 93% of the soil samples were positive for at least one Bcc genomovar, depending on the PCR assay system used. Cloning and sequencing were performed to check the specificity of the PCR assays. Sequence analysis of the 463-bp 16S rRNA inserts from eight clones indicated that all were from members of the Bcc. The four soil samples from which these clones were generated did not yield isolates identified as Bcc. Based on PCR detection, Bcc appears to be prevalent in soil from urban and suburban environments. Culture-based recovery of Bcc may underestimate environmental populations.