Diurnal effects on sporangium and zoospore production by Phytophthora ramorum on Rhododendron 'Cunningham's White'.

We evaluated sporangium and zoospore production by three isolates of Phytophthora ramorum on Rhododendron 'Cunningham's White' leaves under light and dark conditions at both variable and constant (14 C) temperature. P. ramorum-infected leaves were detached and placed in funnels inside of a 62-L plastic storage container located in a growth chamber. Cool mist was introduced to the container to create a high-humidity environment. Sporangia and zoospores were collected over a 4-day period by misting leaves with 5 mL of distilled water, which was collected in conical test tubes that also contained runoff from the misting. Spores were collected daily just before a 13-h light period and again just before an 11-h dark period. Sporangia and zoospores in the collection tubes were counted using a dissecting microscope following staining with lactoglycerin/aniline blue. Large differences in sporangium and zoospore numbers observed for the dark versus light periods were observed on days 2, 3, and 4. A diurnal effect has been observed for production of propagules of other oomycetes, but such effects have not been previously reported for P. ramorum. This information will help provide a better understanding of patterns of inoculum production by P. ramorum and resulting fluctuations in inoculum density that will influence sudden oak death epidemics in forest ecosystems in the United States and other countries where it occurs.

Population Structure of Phytophthora plurivora on Rhododendron in Oregon Nurseries.

Phytophthora plurivora is a recently described plant pathogen, formerly recognized as P. citricola. Recent sampling of Pacific Northwest nurseries frequently encountered this pathogen, and it has been shown to be among the most damaging Phytophthora pathogens on ornamentals. We characterized the population structure of P. plurivora in a survey of four Oregon nurseries across three different counties with focus on Rhododendron hosts. Isolates were identified to the species level by Sanger sequencing and/or a PCR-RFLP assay of the internal transcribed spacer (ITS) region. We used genotyping-by-sequencing to determine genetic diversity. Variants were called de novo, resulting in 284 high-quality variants for 61 isolates after stringent filtering. Based on Fst and AMOVA, populations were moderately differentiated among nurseries. Overall, population structure suggested presence of one dominant clonal lineage in all nurseries, as well as isolates of cryptic diversity mostly found in one nursery. Within the clonal lineage, there was a broad range of sensitivity to mefenoxam and phosphorous acid. Sensitivity of the two fungicides was correlated. P. plurivora was previously assumed to spread clonally, and the low genotypic diversity observed within and among isolates corroborated this hypothesis. The broad range of fungicide sensitivity within the P. plurivora population found in PNW nurseries has implications for managing disease caused by this important nursery pathogen. These findings provide the first perspective into P. plurivora population structure and phenotypic plasticity in Pacific Northwest nurseries.

The Effect of Leaf Wetness on Phytophthora ramorum Zoospore Infection of Rhododendron 'Cunningham's White' and Viburnum tinus.

We performed studies using zoospore inoculum combined from nine isolates of Phytophthora ramorum and determined the effect of leaf wetness on infection of whole plants of Rhododendron 'Cunningham's White' and Viburnum tinus. The mean percentage of infected leaves for both host species increased gradually across a dew chamber moisture period of 1 to 6 h, reaching approximately 80% infection by 6 h. We also evaluated the effect of a postinoculation drying period on infectivity of the two host species with zoospore inoculum. With a 30-min postinoculation drying period, Rhododendron 'Cunningham's White' sustained less than 40% infected leaves, whereas V. tinus had an infection rate of almost 75% infected leaves. Disease percentages for both host species declined sharply with drying periods longer than 30 min. Knowledge of infectivity parameters for P. ramorum will provide a better understanding of epidemic development and lead to improved recommendations for control.

Variation in Disease Severity Caused by Phytophthora cinnamomi, P. plurivora, and Pythium cryptoirregulare on Two Rhododendron Cultivars.

Rhododendrons are an important crop in the ornamental nursery industry, but are prone to Phytophthora root rot. Phytophthora root rot is a continuing issue on rhododendrons despite decades of research. Several Phytophthora species are known to cause root rot, but most research has focused on P. cinnamomi, and comparative information on pathogenicity is limited for other commonly encountered oomycetes, including Phytophthora plurivora and Pythium cryptoirregulare. In this study, three isolates each of P. cinnamomi, P. plurivora, and Py. cryptoirregulare were used to inoculate rhododendron cultivars Cunningham's White and Yaku Princess at two different inoculum levels. All three species caused disease, especially at the higher inoculum level. P. cinnamomi and P. plurivora were the most aggressive pathogens, causing severe root rot, whereas Py. cryptoirregulare was a weak pathogen that only caused mild disease. Within each pathogen species, isolate had no influence on disease. Both P. cinnamomi and P. plurivora caused more severe disease on Cunningham's White than on Yaku Princess, suggesting that the relative resistance and susceptibility among rhododendron cultivars might be similar for both pathogens. Reisolation of P. cinnamomi and P. plurivora was also greater from plants exhibiting aboveground symptoms of wilting and plant death and belowground symptoms of root rot than from those without symptoms. Results show that both P. cinnamomi and P. plurivora, but not Py. cryptoirregulare, are important pathogens causing severe root rot in rhododendron. This study establishes the risks for disease resulting from low and high levels of inoculum for each pathogen. Further research is needed to evaluate longer term risks associated with low inoculum levels on rhododendron health and to explore whether differences among pathogen species affect disease control.

Effects of Phytophthora ramorum on volatile organic compound emissions of Rhododendron using gas chromatography-mass spectrometry.

Phytophthora ramorum is an invasive and devastating plant pathogen that causes sudden oak death in coastal forests in the western United States and ramorum blight in nursery ornamentals and native plants in various landscapes. As a broad host-range quarantine pest that can be asymptomatic in some hosts, P. ramorum presents significant challenges for regulatory efforts to detect and contain it, particularly in commercial nurseries. As part of a program to develop new detection methods for cryptic infections in nursery stock, we compared volatile emissions of P. ramorum-inoculated and noninoculated Rhododendron plants using three gas chromatography-mass spectrometry methods. The first used a branch enclosure combined with headspace sorptive extraction to measure plant volatiles in situ. Seventy-eight compounds were found in the general Rhododendron profile. The volatile profile of inoculated but asymptomatic plants (121 days post-inoculation) was distinguishable from the profile of the noninoculated controls. Three compounds were less abundant in inoculated Rhododendron plants relative to noninoculated and mock-inoculated control plants. A second method employed stir bar sorptive extraction to measure volatiles in vitro from leaf extractions in methanol; 114 volatiles were found in the overall profile with 30 compounds less abundant and one compound more abundant in inoculated Rhododendron plants relative to mock-inoculated plants. At 128 days post-inoculation, plants were asymptomatic and similar in appearance to the noninoculated controls, but their chemical profiles were different. In a third technique, volatiles from water runoff from the soil of potted healthy and inoculated Rhododendron plants were compared. Runoff from the inoculated plants contained four unique volatile compounds that never appeared in the runoff from mock-inoculated plants. These three volatile detection techniques could lead to innovative approaches that augment detection and diagnosis of P. ramorum and oomycete pathogens in nurseries and other settings. Graphical abstract Detection of volatile signatures may aid in discriminating healthy vs. infected but asymptomatic plants in nursery and greenhouse facilities.

Host plants of Empria sawflies (Hymenoptera, Tenthredinidae) in Japan include Rhododendron (Ericaceae).

New host plant records are given for six Empria species from Japan. They are Rosa multiflora [Rosaceae] for E. honshuana Prous & Heidemaa, 2011, Rubus sp. [Rosaceae] for E. japonica Heidemaa & Prous, 2011, Geum japonicum and G. calthifolium var. nipponicum [Rosaceae] for E. loktini Ermolenko, 1971, Rosa multiflora, Potentilla indica and probably Rubus parvifolius [Rosaceae] for E. quadrimaculata Takeuchi, 1952, Rhododendron molle subsp. japonicum [Ericaceae] for E. takeuchii Prous & Heidemaa, 2011, and Geum japonicum and Filipendula camtschatica [Rosaceae] for E. tridentis Lee & Ryu, 1996. This is the first record of Ericaceae as a host plant of Empria. The mode of host shifts in the evolution of Empria is inferred by using a phylogenetic hypothesis proposed by Prous et al. (2011a).

A new genus of Macrosiphini Wilson, 1910 (1887) (Hemiptera: Aphididae) from Rhododendron in Turkey.

Rhododendraphis tuatayae gen. n., sp. n. living on Rhododendron sp. (Ericaceae) in Turkey is described based on apterous viviparous females. The new genus is morphologically most similar to Rostratusaphis Fang and Qiao, 2009, but differs in several important characters. Differences between the new genus and other aphid genera with a spinulose second tarsal segment living on Rhododendron spp. worldwide are described. Type specimens are deposited at the Natural History Museum of London (BMNH).

CLIMATE CONDITIONS AFFECTING THE WITHIN-PLANT SPREAD OF BROAD MITES ON AZALEA.

The broad mite Polyphagotarsonemus latus (Banks) is considered a major pest in potted azalea, Flanders' flagship ornamental crop of Rhododendron simsii hybrids. In addition to severe economic damage, the broad mite is dreaded for its increasing resistance to acaricides. Due to restrictions in the use of broad spectrum acaricides, Belgian azalea growers are left with only three compounds, belonging to two mode of action groups and restricted in their number of applications, for broad mite control: abamectin, milbemectin and pyrethrin. Although P. latus can be controlled with predatory mites, the high cost of this system makes it (not yet) feasible for integration into standard azalea pest management systems. Hence, a maximum efficacy of treatments with available compounds is essential. Because abamectin, milbemectin and pyrethrin are contact acaricides with limited trans laminar flow, only broad mites located on shoot tips of azalea plants will be controlled after spraying. Consequently, the efficacy of chemical treatments is influenced by the location and spread of P. latus on the plant. Unfortunately, little is known on broad mites' within-plant spread or how it is affected by climatic conditions like temperature and relative humidity. Therefore, experiments were set up to verify whether climate conditions have an effect on the location and migration of broad mites on azalea. Broad mite infected azalea plants were placed in standard growth chambers under different temperature (T:2.5-25°C) and relative humidity (RH:55-80%) treatments. Within-plant spread was determined by counting mites on the shoot tips and inner leaves of azalea plants. Results indicate that temperature and relative humidity have no significant effect on the within-plant spread of P. latus. To formulate recommendations for optimal spray conditions to maximize the efficacy of broad mite control with acaricides, further experiments on the effect of light intensity and rain are scheduled.

Phytophthora kernoviae oospore maturity, germination, and infection.

Limited information is known on the basic biology of the recently described Phytophthora kernoviae that produces homothallic oospores. In this study, different P. kernoviae isolates were used to investigate oospore maturity, germination, and infection. All isolates produced oospores in V8 broth at 20°C in the dark by 6d. Oospores also formed at 10 and 15°C, but did not form at 25 and 28°C. Continuous light inhibited oospore production of some isolates but had no negative effect on others. Maturation time of the oospores, as noted by germination and staining with tetrazolium bromide, was not much different among the isolates between 2 and 14 weeks. Oospore germination was optimal at 18 and 20°C, and did not occur at 5, 25, and 30°C. Oospore germination under continuous light was higher than in the dark, but individual isolates showed variable results. Rhododendron leaf disks inoculated with oospores and maintained in the dark at 20°C were necrotic after 1 week, while those kept under continuous light did not develop necrosis. The percentage of leaf disks infected with P. kernoviae was lower in the leaves exposed to continuous light (40%) compared to those kept in the dark (100%).

Testate amoebae (Arcellinida and Euglyphida) vs. Ericoid mycorrhizal and DSE fungi: a possible novel interaction in the mycorrhizosphere of ericaceous plants?

Common occurrence of testate amoebae (TA) in the rhizosphere of mycorrhizal plants indicates existence of yet undocumented ecological interactions, involving three distinct groups of organisms: soil protists, mycorrhizal fungi, and their host plants. This tripartite relationship was to date investigated only to a limited extent, despite its probable importance for processes taking place in the mycorrhizosphere. In this study, we (1) explored spectra of different TA genera naturally associated with the rhizoplane of three autochthonous European Rhododendron species, (2) screened natural fungal colonization of the TA shells occupying the rhizoplane of selected rhododendrons, and (3) carried out two in vitro experiments addressing the question whether TA shells may serve as a nutrient source for ericoid mycorrhizal fungi (ErMF) and dark septate endophytes (DSE). Our field observations indicated that TA regularly associated with the rhizoplane of all screened rhododendrons and that ErMF and/or DSE associated with their roots possibly exploited the TA shells as a nutrient source. We were unable to detect any major differences among the TA spectra from the rhizoplanes with respect to the three Rhododendron species. The spectra were dominated by Diplochlamys, Centropyxis, Cyclopyxis, Euglypha, Trinema, and Assulina. Positive, neutral, and negative associations were found for various TA genera x Rhododendron species combinations. The highest fungal colonization was observed in Centropyxidae and Trigonopyxidae, reaching up to 45% of the shells in the case of Trigonopyxis. In the in vitro experiments, both ErMF Rhizoscyphus ericae and DSE Phialocephala fortinii regularly colonized TA shells, utilizing them as a source of nutrients. We hypothesize a complex relationship between ErMF-DSE and TA. If corroborated, it would represent an interesting nutrient loop in the mycorrhizosphere of ericaceous plants.

Rapid, high-throughput detection of azalea lace bug (Hemiptera: Tingidae) predation by Chrysoperla rufilabris (Neuroptera: Chrysopidae), using fluorescent-polymerase chain reaction primers.

Azalea lace bugs, Stephanitis pyrioides (Scott) (Hemiptera: Tingidae), are the most common pest of azaleas (Rhododendron spp.) in nursery production and the landscape. Although pesticides are commonly used to control lace bugs, natural enemies can be a significant source of lace bug mortality. Lacewings (Neuroptera: Chrysopidae) are natural enemies of lace bugs and easily consume them in laboratory studies. Field studies on lacewing biocontrol of azalea lace bugs are underway; however, monitoring lacewing predation in a nursery environment by direct observation is impractical. Here, we describe a fluorescent-polymerase chain reaction method to estimate S. pyrioides consumption based on the gut contents of lacewing predators. Lace bug DNA was detected in fed lacewings up to 32 h after ingestion. More than 80% of the ingested lace bugs were detected using our method with only one false positive result. The assay is both high-throughput and relatively inexpensive, making it a practical approach to documenting lace bug predation in the field.