Elizabeth J. Fichtner

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.

Forest type influences transmission of Phytophthora ramorum in California oak woodlands.

The transmission ecology of Phytophthora ramorum from bay laurel (Umbellularia californica) leaves was compared between mixed-evergreen and redwood forest types throughout winter and summer disease cycles in central, coastal California. In a preliminary multisite study, we found that abscission rates of infected leaves were higher at mixed-evergreen sites. In addition, final infection counts were slightly higher at mixed-evergreen sites or not significantly different than at redwood sites, in part due to competition from other foliar pathogens at redwood sites. In a subsequent, detailed study of paired sites where P. ramorum was the main foliar pathogen, summer survival of P. ramorum in bay laurel leaves was lower in mixed-evergreen forest due to lower recovery from infected attached leaves and higher abscission rates of infected leaves. Onset of inoculum production and new infections of bay laurel leaves occurred later in mixed-evergreen forest. Mean inoculum levels in rainwater and final infection counts on leaves were higher in redwood forest. Based on these two studies, lower summer survival of reservoir inoculum in bay laurel leaves in mixed-evergreen forest may result in delayed onset of both inoculum production and new infections, leading to slower disease progress in the early rainy season compared with redwood forest. Although final infection counts also will depend on other foliar pathogens and disease history, in sites where P. ramorum is the main foliar pathogen, these transmission patterns suggest higher rates of disease spread in redwood forests during rainy seasons of short or average length.

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.

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.

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.

First Report of Lasiodiplodia citricola and Neoscytalidium dimidiatum Causing Death of Graft Union of English Walnut in California

California produces 99% of the English walnuts (Juglans regia) in the USA. In August 2012 in Tulare County, about 5,000 out of 90,000 trees were killed in a walnut nursery by a distinct black canker that developed around the graft union. The cankers appeared to be initiated at the heading cut on the rootstock, and expanded down to the rootstock and through the budded union up to the scion, resulting in mortality of scion. The walnut nursery was located adjacent to a mature walnut orchard. The fungi isolated from the cankers were identified as Lasiodiplodia citricola and Neoscytalidium dimidiatum based on morphological characteristics and DNA sequence comparisons. L. citricola was isolated from one of the 10 graft unions, while N. dimidiatum from the other nine. L. citricola isolates were characterized by white, aerial mycelium on potato dextrose agar that turned gray after 4 days and produced ellipsoidal to ovoid hyaline one-celled conidia that became 2-celled and brown with thick walls and longitudinal striations in the wall (1). N. dimidiatum isolates were characterized by ellipsoid to ovoid, hyaline conidia with a truncate base and an acutely rounded apex, initially 1-celled, and some becoming brown and 2-celled at maturity; no muriform conidia were produced (3). These identifications were confirmed by analyses of the ITS, BT2, and TEF-1α gene regions. The three gene regions were amplified by using the primers and methods described in (4). For L. citricola (isolates 7E78 to 7E80), a DNA sequence BLASTn at GenBank showed 100% identity with accessions GU945354 (ITS) and GU945340 (TEF-1α) of the ex-type specimen (CBS124707, BT2 sequencing data was not available) (3). For N. dimidiatum (isolates 7E61 to 7E63), a BLASTn search showed a high identify (ITS, 100%; BT2, 99%; TEF-1α, 99%) with reference sequence of N. dimidiatum (ITS, GQ330903; BT2, GU251768; TEF-1α, GU251240). Sequences of the studied DNA regions were deposited to GenBank as KC357298 to KC357303 (ITS); KC357304 to KC357309 (BT2); and, KC357310 to KC357315 (TEF-1α). The pathogenicity of L. citricola in comparison with N. dimidiatum in J. regia cvs. Chandler, Tulare, and Vina was evaluated in an orchard at KARE, by using two isolates each of L. citricola (7E78, 7E80) and N. dimidiatum (7E61, 7E63). Pathogenicity tests were performed by inoculating ten 2-year-old branches per isolate in late September 2012 by the method described in (2). After 3 weeks, the average lesion lengths caused by L. citricola on Chandler, Tulare, and Vina were 152, 156, and 188 mm, respectively, and 32, 38, and 34 mm, respectively, caused by N. dimidiatum. The lesion length averages produced on the three cultivars by the four isolates were all significantly (P < 0.05) longer than their respective controls (average length 10 mm on all cultivars). L. citricola, but not N. dimidiatum, killed branches of Chandler, Tulare, and Vina in 10 days. Both L. citricola and N. dimidiatum were reisolated from the inoculated branches and no fungus was isolated from the control, confirming Kochs postulates. These results confirmed that the walnut graft union canker was caused by either L. citricola or N. dimidiatum. To our knowledge, this is the first report of death of newly grafted walnut trees caused by L. citricola and N. dimidiatum worldwide, and also the first report of L. citricola infecting walnut worldwide. References: (1) J. Abdollahzadeh et al. Persoonia. 25:1, 2010. (2) S. F. Chen et al. Plant Dis. 97:994, 2013. (3) D. Pavlic et al. Mycologia. 106:851, 2008. (4) B. Slippers et al. Mycologia. 96:83, 2004.