First Report of Geosmithia morbida on English Walnut and its Paradox Rootstock in California.

Geosmithia morbida, the causal agent of thousand cankers disease (TCD), is vectored by the walnut twig beetle (WTB), Pityophthorus juglandis, causing decline in eastern black walnut, Juglans nigra (4), and canker development on many Juglans species (5). In the summer of 2012, a survey for TCD incidence in English walnut, J. regia, in orchards in California identified many trees with WTB activity and characteristic TCD symptoms. Both the J. regia scion and its Paradox hybrid rootstock (J. hindsii× J. regia) were affected. In some cases, trees exhibited bleeding on the bark surface from WTB entrance holes. Removal of the outer bark revealed cankers in the phloem around the WTB galleries. Two samples were taken from scions and three samples were collected from rootstocks of trees in orchards in northern California. Pieces (~3 to 4 mm2) of symptomatic tissue were placed in acidified potato dextrose agar (APDA), and the plates were incubated for 4 to 5 days at 30°C. Samples exhibiting fungal growth similar in morphology to G. morbida were transferred to PDA plates to obtain pure cultures and then processed to obtain single-spore cultures. Culture morphology for five single-spore isolates (Gm103, Gm104, Gm105, Gm107, and Gm108) was similar to that described by Kolarík et al. (4) for G. morbida. Conidiophores were penicillate and verrucose. Conidia were narrowly cylindrical, 5.2 ± 0.06 × 2.2 ± 0.04 µm (n = 50). Single-spore isolates were then grown in 1% yeast extract glucose liquid culture for 7 to 10 days. DNA was extracted and the ITS region was amplified, including the 5.8S region by using primers ITS1F/ITS4. Sequences were assembled and deposited in GenBank under accessions KJ664793 to KJ664797. Sequences were compared to those in GenBank; all sequences matched (99 to 100% identity) the ITS sequences of G. morbida strain CBS 124663. Pathogenicity tests were performed on 28-cm-long detached branches of J. regia. Four branches per isolate were inoculated with a 5-mm-diameter mycelial plug from a 2-week-old culture. Branches were incubated at room temperature (23 ± 2°C) in a humidified container for 3 weeks, and then canker lengths were measured. Pieces of the cankered area were placed in APDA and incubated as described above with G. morbida re-isolated from the cankers for all of the isolates, completing Koch's postulates. Average canker lengths ranged from 48.6 ± 4.3 to 72.1 ± 7.1 mm. Re-isolated G. morbida exhibited the same growth and reproductive structure morphology in culture on PDA as the original cultures. TCD in association with WTB has been observed in California English walnut orchards since 2008 (1,2,3). However, this is the first report for completion of Koch's postulates and morphological and molecular confirmation of G. morbida in J. regia and the Paradox rootstock, the predominant rootstock used in commercial orchards. TCD is a concern to the walnut industry in California with over 245,000 bearing acres reported in 2012. References: (1) M. Flint et al. CAPCA Adviser 8:36, 2010. (2) A. D. Graves et al. Walnut Twig Beetle and Thousand Cankers Disease: Field Identification Guide. UC-IPM website publication, http://www.ipm.ucdavis.edu/PDF/MISC/thousand_cankers_field_guide.pdf , 2009. (3) J. Hasey et al. (Abstr.) Phytopathology 100:S48, 2010. (4) M. Kolarík et al. Mycologia 103:325, 2011. (5) C. Utley et al. Plant Dis. 97:601, 2013.

Root Infections May Challenge Management of Invasive Phytophthora spp. in U.K. Woodlands.

Because sporulation of Phytophthora ramorum and P. kernoviae on Rhododendron ponticum, an invasive plant, serves as primary inoculum for trunk infections on trees, R. ponticum clearance from pathogen-infested woodlands is pivotal to inoculum management. The efficacy of clearance for long-term disease management is unknown, in part due to lack of knowledge of pathogen persistence in roots and emerging seedlings. The main objectives of this work were to (i) investigate whether both pathogens infect R. ponticum roots, (ii) determine the potential for residual inoculum of P. kernoviae to infect R. ponticum seedlings in cleared woodlands, and (iii) assess potential for R. ponticum roots to support survival and transmission of P. kernoviae. Roots of R. ponticum were collected from both unmanaged and cleared woodlands and assessed for pathogen recovery. Both P. ramorum and P. kernoviae were recovered from asymptomatic roots of R. ponticum in unmanaged woodlands, and P. kernoviae was recovered from asymptomatic roots from seedlings in cleared woodland. Oospore production of P. kernoviae was observed in naturally infected R. ponticum foliage and in inoculated roots. Roots also supported P. kernoviae sporangia production. The results of this study suggest that post-clearance management of R. ponticum regrowth is necessary for long-term inoculum management in invaded woodlands.

Survival, dispersal, and potential soil-mediated suppression of Phytophthora ramorum in a California redwood-tanoak forest.

Because the role of soil inoculum of Phytophthora ramorum in the sudden oak death disease cycle is not well understood, this work addresses survival, chlamydospore production, pathogen suppression, and splash dispersal of the pathogen in infested forest soils. Colonized rhododendron and bay laurel leaf disks were placed in mesh sachets before transfer to the field in January 2005 and 2006. Sachets were placed under tanoak, bay laurel, and redwood at three vertical locations: leaf litter surface, litter-soil interface, and below the soil surface. Sachets were retrieved after 4, 8, 20, and 49 weeks. Pathogen survival was higher in rhododendron leaf tissue than in bay tissue, with >80% survival observed in rhododendron tissue after 49 weeks in the field. Chlamydospore production was determined by clearing infected tissue in KOH. Moist redwood-associated soils suppressed chlamydospore production. Rain events splashed inoculum as high as 30 cm from the soil surface, inciting aerial infection of bay laurel and tanoak. Leaf litter may provide an incomplete barrier to splash dispersal. This 2-year study illustrates annual P. ramorum survival in soil and the suppressive nature of redwood-associated soils to chlamydospore production. Infested soil may serve as primary inoculum for foliar infections by splash dispersal during rain events.

Disease Risk of Potting Media Infested with Phytophthora ramorum Under Nursery Conditions.

Phytophthora ramorum has been found in potting media of containerized plants; however, the role of infested media on disease development under nursery conditions is unknown. This study assesses pathogen survival, sporulation, and infectivity to rhododendron plants in nursery pots with infected leaf litter that were maintained under greenhouse and field conditions. The influence of environmental conditions and irrigation method on disease incidence was also assessed. Infected leaf disks were buried below the soil surface of potted rhododendrons and retrieved at approximately 10-week intervals for up to 66 weeks. Pathogen survival was assessed by either isolation or induction of sporulation in water over three experimental periods. P. ramorum was recovered from infected leaf disks incubated in planted pots for longer than 1 year. Chlamydospores and sporangia formed on hydrated leaf disks but relative production of each spore type varied with the duration of incubation in soil. Root infections were detected after 40 weeks in infested soil. Foliar infections developed on lower leaves but only after spring rain events. Sprinkler irrigation promoted the development of foliar infections; no disease incidence was observed in drip-irrigated plants unless foliage was in direct contact with infested soil. Management implications are discussed.

Detection, Distribution, Sporulation, and Survival of Phytophthora ramorum in a California Redwood-Tanoak Forest Soil.

ABSTRACT Recovery of Phytophthora ramorum from soils throughout sudden oak death-affected regions of California illustrates that soil may serve as an inoculum reservoir, but the role of soil inoculum in the disease cycle is unknown. This study addresses the efficacy of soil baiting, seasonal pathogen distribution under several epidemiologically important host species, summer survival and chlamydospore production in soil, and the impact of soil drying on pathogen survival. The efficacy of rhododendron leaves and pears as baits for detection of soilborne propagules were compared. Natural inoculum associated with bay laurel (Umbellularia californica), tanoak (Lithocarpus densiflorus), and redwood (Sequoia sempervirens) were determined by monthly baiting. Summer survival and chlamydospore production were assessed in infected rhododendron leaf disks incubated under bay laurel, tanoak, and redwood at either the surface, the litter/soil interface, or in soil. Rhododendron leaf baits were superior to pear baits for sporangia detection, but neither bait detected chlamydospores. Most inoculum was associated with bay laurel and recovery was higher in soil than litter. Soil-incubated inoculum exhibited over 60% survival at the end of summer and also supported elevated chlamydospore production. P. ramorum survives and produces chlamydospores in forest soils over summer, providing a possible inoculum reservoir at the onset of the fall disease cycle.

Differential Sensitivity of Phytophthora parasitica var. nicotianae and Thielaviopsis basicola to Monomeric Aluminum Species.

ABSTRACT Aluminum (Al) is toxic to many plant pathogens, including Thielaviopsis basicola and Phytophthora parasitica var. nicotianae. Because fungi-toxicity of Al has been described in soils over a wide pH range, multiple species of Al may be responsible for pathogen suppression. The goals of this work were to determine the sensitivity of T. basicola and P. para-sitica var. nicotianae to Al over a range of pH values, quantify the toxicity of monomeric Al species to production of sporangia of P. parasitica var. nicotianae and chlamydospores of T. basicola, and detect the accumulation of Al in pathogen structures. A complete factorial treatment design was used with Al levels ranging from 0 to 100 muM and pH levels ranging from 4 to 6 in a minimal salts medium. The chemistry of test solutions was modeled using GEOCHEM-PC. Colonies were grown in 5% carrot broth, and after 1 or 2 days, the nutrient solution was removed, colonies were rinsed with water, and Al test solutions were added to each of four replicate plates. After 2 days, propagules were counted and colonies were stained with the Al-specific, fluorescent stain lumogallion. The oomycete P. parasitica var. nicotianae was sensitive to multiple monomeric Al species, whereas sensitivity of T. basicola to Al was pH-dependent, suggesting that only Al(3+) is responsible for suppression of this fungal pathogen. Chlamydospore production by T. basicola was inhibited at pH values <5.0 and Al levels >20 muM, whereas sporangia production by P. parasitica was inhibited at Al levels as low as 2 muM across all pH values tested. The lumogallion stain was an effective technique for detection of Al in fungal tissues. Aluminum accumulated in sporangia and zoospores of P. parasitica var. nicotianae and in nonmelanized chlamy-dospores of T. basicola, but not in cell walls of either organism. The differential sensitivity of the two organisms may indicate that true fungi respond differently to Al than members of the oomycota, which are more closely related to plants.

Nonphytotoxic Aluminum-Peat Complexes Suppress Phytophthora parasitica.

ABSTRACT Amendment of peat-based potting media with Al(2)(SO(4))(3) suppresses damping-off of Vinca (Catharanthus roseus) caused by Phytophthora parasitica. The species of aluminum (Al) responsible for disease suppression have not been identified. The objective of this study was to determine the effects of amount and pH of Al(2)(SO(4))(3) amendment solutions on survival of P. parasitica. In separate experiments, peat was amended with Al(2)(SO(4))(3) solutions adjusted to pH 4 or 6 at either 0.0158 or 0.0079 g of Al per gram of peat. Amended peat was placed in Büchner funnels maintained at -2.5 kPa matric potential. Peat was infested with P. parasitica by placing zero, two, or five colonized Vinca leaf disks in each funnel, and 15 Vinca seeds were placed in each funnel. After 24 h, the matric potential was brought to 0 kPa to induce zoospore release and returned to -2.5 kPa after 24 h. Pathogen populations and stand counts were assessed after 2-week incubation. Al amendment solutions at both pH 4 and 6 reduced pathogen populations at 0.0158 g of Al per gram of peat. Solutions at pH 4 reduced pathogen populations by more than 90% at both inoculum levels; amendment solutions at pH 6 reduced populations by 95% at the low inoculum level and 65% at the high inoculum level. The prevalence of Al(OH)(2)(+) in peat amended with Al(2)(SO(4))(3) solution at pH 6 suggests that ions other than Al(3+) may be responsible for pathogen suppression. Based on the difference in chemical conditions of Al-amended peat and suppressive mineral soils, the mechanism of Al-mediated suppression of plant pathogens is speculated to be different in the two systems. Peat containing Al-peat complexes was chemically suppressive to P. parasitica and may confer Al-mediated suppression of plant pathogens with a nonphytotoxic form of Al.

Abiotic and Biological Suppression of Phytophthora parasitica in a Horticultural Medium Containing Composted Swine Waste.

ABSTRACT Horticultural potting media have been amended with compost to enhance biological suppression and with Al(2)(SO(4))(3) to enhance abiotic suppression of plant pathogens, but these factors have not been simultaneously incorporated into the same medium. In this study, the efficacy of aluminum (Al)-amended potting medium containing 20% composted swine waste (CSW) was assessed for control of Phytophthora parasitica (syn. P. nicotianae), a soilborne pathogen causing damping-off of many horticultural bedding plants. Steamed and unsteamed media were amended with no Al or Al at 0.0079 g of Al g(-1) of medium with an Al(2)(SO(4))(3) solution at either pH 4 or pH 6. Infested leaf disks were buried for 2-day durations beginning 0, 6, 13, and 21 days after Al amendment. The number of sporangia produced on infested leaf disks was assessed. A similar experiment was conducted to determine the effect of steaming and Al amendments on pathogen populations. Medium treated with the pH 4 solution consistently reduced sporangia production between 38 and 65% on day 0, but no Al effect was noted at subsequent time points. The pH 6 amendment did not consistently affect sporangia production. Exchangeable Al levels decreased over time, and abiotic suppression was only observed at >2 muM Al g(-1) of medium. Pathogen populations were occasionally affected by steaming and Al. Sporangia production in unsteamed medium was reduced by 50% on leaf disks buried on days 6, 13, and 21, but not on day 0. Al amendment of a 20% CSW potting medium enhanced suppression of P. parasitica and abiotic suppression occurred before biological suppression developed.