Cunningham's White Research Articles

Clonal micropropagation of rhododendrons as affected by varietal characteristics and nutrient media

Purpose. To develop clonal micropropagation for representatives of the genus Rhododendron L. for different varietal characteristics and nutrient media.
 Methods. Rhododendron seeds of various cultivars were used for sterilization and in vitro culture: 'Summetglut', 'Cunningham's White', 'Grandiflorum', 'Libretto', 'Yakushymanskyi', 'Shamrock' and 'Balalaika'. Sterile shoots were planted on different types of nutrient media, such as Murashige and Skoog (MS), Hamborg and Evelegh (B5) and Anderson (An), modified with different compositions and concentrations of hormones BAP and zeatin.
 Results. Regardless of varietal characteristics, the largest number (12) and height (7 cm) of rhododendron shoots was formed on the Anderson medium. The smallest number and height of shoots were noted in cultivars 'Yakushymanskyi' and 'Balalaika' on all nutrient media. On the Anderson nutrient medium, variety 'Cunningham's White' formed 10 shoots and 'Grandiflorum' 12 shoots. These varieties also had the highest height of 7 cm. On the MS medium, these varieties formed 2 and 4 shoots of 2.5 and 3 cm high, respectively. The best shoot-forming ability was recorded on the Anderson nutrient medium, with 8 to 22 shoots. The largest number (20 and 22) of newly formed shoots was noted in variety 'Grandiflorum', 20 and 22 shoots, respectively. This number of shoots was somewhat lower in varieties 'Shamrock' (17 and 21), 'Cunningham's White' (18), 'Libretto' (15 and 17), 'Yakushymanskyi' (8 and 10) and 'Balalaika' (9).
 Conclusions. With the use of zeatin, the number of shoots in all studied cultivars was almost the same as with the BAP concentration of 1.0 mg/l. The lowest number and the lowest height of shoots were formed on the Hamborg and Eveleg nutrient medium, besides, the shoots of all varieties were in suppressed condition. This medium and its modifications do not provide a sufficient number of shoots and cannot be recommended for clonal micropropagation of rhododendrons. MS nutrient medium with the addition of BAP (1.0 mg/l) makes it possible to obtain cultivars from 0.8 to 7 cm in height and with the use of zeatin (0.5 mg/l) from 1.8 to 9 cm, respectively. Regardless of varietal characteristics and nutrient media, vitrification of shoots was observed with an increase in the concentration of zeatin to 1.0 mg/l.

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.

Aster Yellows Subgroup 16SrI‐C Phytoplasma in Rhododendron hybridum

AbstractSymptoms of unknown aetiology on Rhododendron hybridum cv. Cunningham's White were observed in the Czech Republic in 2010. The infected plant had malformed leaves, with irregular shaped edges, mosaic, leaf tip necrosis and multiple axillary shoots with smaller leaves. Transmission electron microscopy showed phytoplasma‐like bodies in phloem cells of the symptomatic plant. Phytoplasma presence was confirmed by polymerase chain reaction using phytoplasma‐specific, universal and group‐specific primer pairs. Restriction fragment length polymorphism analysis of 16S rDNA enabled classification of the detected phytoplasma into the aster yellows subgroup I‐C. Sequence analysis of the 16S‐23S ribosomal operon of the amplified phytoplasma genome from the infected rhododendron plant (1724 bp) confirmed the closest relationship with the Czech Echinacea purpurea phyllody phytoplasma. These data suggest Rhododendron hybridum is a new host for the aster yellows phytoplasma subgroup 16SrI‐C in the Czech Republic and worldwide.

First Report of Calonectria Leaf Spot Caused by Calonectria colhounii (Anamorph Cylindrocladium colhounii) on Rhododendron in Belgium.

Belgium is one of the most important Rhododendron-producing areas in Europe, with an annual sale of approximately 1.6 million plants. In June 2010, an outbreak of leaf spots on several thousands of Rhododendron cv. Marcel Menard plants took place at a nursery near Gent. Diseased plants showed dark brown leaf spots that enlarged and finally resulted in leaf drop. Symptoms developed most explicitly on this cultivar, especially after standard repotting during May or June and when repotting was followed by a few days of unusually warm temperatures (30 to 35°C). The leading edge of diseased leaf tissue was excised, surface disinfected with 1% NaOCl for 60 s, and rinsed twice with sterile distilled water before being plated onto potato dextrose agar (PDA). After 5 days of incubation at 21°C in the dark, Cylindrocladium-like fungal colonies with white aerial mycelium and amber-brown growth within the agar consistently developed. Mycelium was transferred aseptically to fresh plates of PDA and incubated for 10 to 14 days at 17°C under a 12-h fluorescent light regimen to study the morphological characteristics. Conidiophores showed a penicillate arrangement of fertile branches, producing two to six phialides. They arose from a stipe and terminated in a clavate vesicle (3 to 5 μm). Conidia were straight, cylindrical, rounded at both ends, three septate, and measured 60 to 70 × 4 to 6 μm. Yellow subglobose to oval perithecia were abundantly produced. Asci were clavate, four spored, and measured 100 to 150 × 15 to 30 μm. Ascospores were hyaline, three septate, and measured 50 to 65 × 5 to 6 μm. These characteristics are consistent with those of Calonectria colhounii Peerally (anamorph Cylindrocladium colhounii) (1). The β-tubulin gene was PCR-amplified with DNA extracted from the mycelium and the T1 and T2 primers (3), sequenced directly with a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA), and the DNA sequence was deposited (GenBank Accession No. JF802784). BLASTn alignment showed 99% identity (525 of 526 nucleotides) with the β-tubulin DNA sequence derived from Calonectria colhounii CBS 293.79 (GenBank Accession No. DQ190564). A spore suspension (105 conidia per ml) was prepared from a 1-week-old culture, and 50-μl drops were used to inoculate the abaxial side of 10 detached 1-year-old leaves from Rhododendron cv. Cunningham's White. Ten control leaves were inoculated with water. The leaves were placed in a moist chamber and incubated at 21°C in the dark. After 5 to 6 days, all spore-inoculated leaves showed lesions identical to those on the naturally infected leaves, while the water-inoculated leaves remained symptom free. Following the original procedure, the fungus was reisolated from the diseased leaves and the morphological characteristics of the resulting culture were the same as those of the inoculated isolate, completing Koch's postulates. This fungus has been described on Rhododendron in the United States (2), but to our knowledge, this is the first record of Calonectria colhounii on Rhododendron in Belgium.

Effects of high concentrations of calcium salts in the substrate and its pH on the growth of selected rhododendron cultivars

For proper growth and development, rhododendrons need acidic soils, whereas calcium carbonate (CaCO<sub>3</sub>) in the substrate markedly limits their growth. In this study, we analysed the reactions of rhododendrons to high concentrations of calcium salts and pH in the substrate. We used 4-month-old seedlings of <em>Rhododendron</em> 'Cunningham's White' and 1.5-year-old seedlings and rooted cuttings of <em>R.</em> 'Cunningham's White' and <em>R.</em> 'Catawbiense Grandiflorum'. Their reactions depended mostly on calcium salt type added to the substrate (sulphate or carbonate). An increase in concentrations of phenolic compounds was detected mostly in roots of the plants grown in a substrate with a high calcium carbonate content. Addition of calcium salts to the substrate caused a significant rise in total nonstructural carbohydrates in leaves and roots of the studied plants. As compared to the control, an increase in substrate pH in the variant with calcium carbonate limited the activity of acid phosphatase, while lowering of substrate pH in the variant with calcium sulphate, significantly increased its activity. Along with the rise in substrate pH, a remarkable increase was observed in the activity of nonspecific dehydrogenase (DHA) in the substrate with CaCO<sub>3</sub>, as compared to the control. Unfavourable soil conditions (high calcium content and alkaline pH) caused a decrease in assimilation of minerals by the studied plants (mostly phosphorus and manganese). Our results show that the major factor limiting rhododendron growth is an increase in substrate pH, rather than an increase in the concentration of calcium ions.

Episodic Abiotic Stress as a Potential Contributing Factor to Onset and Severity of Disease Caused by Phytophthora ramorum in Rhododendron and Viburnum.

Phytophthora ramorum attacks many forest and nursery species, primarily causing trunk or stem cankers, foliar blight, and dieback, and in some species root infection has been demonstrated. However, the abiotic and edaphic factors that influence infection and disease development are unresolved. Root infection by P. ramorum and the potential for mild abiotic stress in disease predisposition was examined with Rhododendron hybrid Cunningham's White and Viburnum tinus cv. Spring Bouquet. To impose water stress in a uniform and synchronous manner, osmotic stress induced with 0.2 M NaCl was selected. Roots were exposed to NaCl for 16 to 24 h in modified hydroponic culture or standard potting soil, removed from the NaCl, and then inoculated with zoospores. In the hydroponic regime, disease symptoms developed in Rhododendron and V. tinus plants within 1 week after inoculation of salt-stressed roots, whereas symptom development was delayed in nonstressed, inoculated plants. Microscopic examination of roots from both species revealed that their apices were covered with sporangia of P. ramorum. On potted Rhododendron plants inoculated by applying zoospores directly to the soil, stem lesions developed rapidly in salt-stressed plants, with death of the plant occurring within 4 weeks after inoculation. Nonstressed plants survived for 6 to 8 weeks before succumbing to disease, and symptom development in these plants was delayed by 1 to 2 weeks relative to the inoculated, salt-stressed plants. A postinfection episode of salt stress to inoculated roots in the hydroponic regime resulted in significantly faster development of stem lesions in Rhododendron relative to nonstressed, inoculated plants.

Susceptibility to Phytophthora ramorum and Inoculum Production Potential of Some Common Eastern Forest Understory Plant Species.

Twenty-five plant species (21 genera, 14 families), which comprise a portion of the understory in forests of the Eastern United States, were evaluated for susceptibility to infection by Phytophthora ramorum. The degree to which P. ramorum is able to form sporangia and chlamydospores was also assessed on these hosts. Seedlings were spray-inoculated with a mixture (4,000 sporangia/ml) of four P. ramorum isolates followed by incubation in a dew chamber at 20°C in darkness for 5 days. Percent infection on individual leaves/leaflets was assessed visually. Mean percent leaf area infected ranged from 0.7% for Smilax rotundifolia to 93.8% for Kalmia latifolia. Eight plant species tested developed significantly larger lesion areas than those found on susceptible control Rhododendron 'Cunningham's White'. Fourteen species in addition to the susceptible control exhibited infection of over 90% of their leaves. Sporangia production by P. ramorum varied considerably among plant species, ranging from 36 per cm2 lesion area on Myrica pennsylvannica to 2,001 per cm2 lesion area on Robinia pseudoacacia. Numbers of chlamydospores produced per 6-mm-diameter leaf disk incubated in a P. ramorum sporangia suspension ranged from 25 on Ilex verticillata to 493 on Rhus typhina. The results indicate that many common understory species in Eastern U.S. forests are susceptible to P. ramorum and capable of providing ample sources of inoculum (sporangia and chlamydospores) for forest epidemics should the pathogen be introduced and should temperature and moisture conditions exist that are conducive to disease development.

Evaluation of Fungicides for the Control of Phytophthora ramorum Infecting Rhododendron, Camellia, Pieris, and Viburnum

Fungicides were evaluated for pre- and post-infection control of ramorum blight, caused by Phytophthora ramorum, on Rhododendron cvs. Cunningham's White and Irish Lace, Camellia japonica, Pieris japonica, and Viburnum tinus. Cyazofamid, dimethomorph, mefenoxam, pyraclostrobin, and fenamidone applied as foliar sprays consistently provided preventative control as indicated by reduced lesion size compared to water controls. These fungicides provided preventative activity for at least 28 days in the tested species except in Rhododendron where fungicides were active for at least 14 days following application. With preventative fungicide applications, the pathogen was recovered from most fungicide-treated leaves by isolation onto selective media. Dimethomorph consistently reduced the percent recovery from diseased Rhododendron leaves. With post-infection treatments, the fungicides did not significantly reduce lesion growth and percent recovery of the pathogen. The pathogen was recovered from lesions consistently at least 6 weeks after fungicide application in Rhododendron regardless of treatment on intact and fallen diseased leaves. However, the cultures resulting from isolations of diseased tissue treated with cyazofamid and dimethomorph were significantly slower growing than those cultures from other fungicide treatments. P. ramorum management issues relating to fungicide use in commercial nurseries are discussed Accepted for publication 12 October 2007. Published 8 February 2008.

The Occurrence of Phytophthora ramorum in Nursery Stock in California, Oregon, and Washington States

Data from nursery surveys conducted in 2003, 2004, and 2005 for ramorum blight and dieback caused by Phytophthora ramorum were analyzed to determine which plant cultivars were likely to be infected with P. ramorum. Susceptible plant host genera, species, and cultivars were identified using culture and nested-polymerase chain reaction analyses, and disease incidence was calculated. For the genus, species, or cultivar analyses at least 20 samples were utilized in this study. The results indicated that the genus Camellia had the highest disease incidence, followed by Rhododendron and Viburnum. Camellia cvs. Setsugekka, Jean May, April Remembered, Winter's Fire, Kramer's Supreme, Bonanza, Magnoliaeflora, Apple Blossom, Silver Waves, Yuletide, and Nuccio's Gem showed the highest risk of P. ramorum infection. Four Rhododendron cvs. Purple Splender, Cunningham's White, Unique, and Nova Zembla were also grouped into the high risk category. All other Rhododendron and Viburnum plant species were at medium or low risk of infection by the pathogen. This study indicated that there is a large variation in disease incidence among genera and specific cultivars within a genus. A series of controlled, replicated, and well designed experiments involving a large sample size of plant cultivars is essential in order to identify the underlying reasons for the observed variation in infection risks of the surveyed data. Accepted for publication 25 January 2006. Published 15 March 2006.

Phytophthora cinnamomi

<i>Phytophthora cinnamomi</i>

082 Infection and Transformation of Rhododendron by Agrobacterium tumefaciens Strain B6

The objective of this study was to determine if selected strains of Agrobacterium could infect microshoots of Rhododendron catawbiense. Fifteen microshoot stems of R. catawbiense var. album `America', `Joe Paterno', and `Cunningham's White' were inoculated with two drops (about 25 μL) of wild type Agrobacterium tumefaciens strains C58 or B6 or with wild type A. rhizogenes strain E8/73. Five control shoots were inoculated with 1.2 mM KH2PO4 buffer. Microshoots were grown on woody plant medium (WPM) supplemented with 4.9 μM 2iP. Six weeks after inoculation galls that formed were excised from the microshoots and placed on WPM that lacked plant growth regulators but contained 300 mg·L-1 cefotaxime. In another study, these wild-type bacterial strains were genetically modified by inserting the pBINm-gfp5-ER plasmid, which contained genes coding for NPTII and green fluorescence protein (GFP), into the bacteria. These modified strains were inoculated on 15 stems of the three rhododendron cultivars and one variety. Calluses that formed were excised, placed on basal WPM with cefotaxime, and allowed to proliferate. Wild type C58 induced galls to form on `Joe Paterno', R.c. album, and `Cunningham's White' stems, whereas wild type B6 caused galls to form only on the latter two types of rhododendron. Wild-type E8/73 failed to induce gall formation on the rhododendrons. Only genetically modified B6 caused galls to form on only `Cunningham's White' microshoots (seven of 15 inoculated stems). Three of these galls fluoresced green under ultraviolet light. Physical presence of the NPTII and GFP genes in the plant genome was determined by polymerase chain reaction. This study demonstrated that R. catawbiense is susceptible to Agrobacterium infection, and this plant can be genetically transformed.

Cold hardiness of diploid and corresponding autotetraploid rhododendrons

SummaryFreezing tolerance was studied in two diploid rhododendron cultivars, Rhododendron `PJM' and R. `Cunningham's White', and their tetraploid derivatives, R. `Northern Starburst' (NSB) and CW4. After the growing season and initial hardening at 15to 108C in a greenhouse, the acclimation regime in a phytotron comprised three consecutive weeks at 15, 11 and 228C. The hardiness was studied with controlled freezing tests before each decrease in temperature and at the end of the experiment. The injury on leaves and stem sections was visually assessed and the data analysed using logit models. Electrolyte leakage tests provided a comparison. Flower bud hardiness was studied by differential thermal analysis. Leaves of the diploid clones attained better cold hardiness than those of the tetraploid clones. At the end of the experiment, the LT50 of the `PJM' leaves was 247.18C and that of NSB 234.28C. The diploid `Cunningham's White' leaves hardened to 235.08C and the tetraploid ones to 222.78C. The LT50 of the stems of `PJM' and NSB was between 2338C and 2408C depending on test and date. There was no statistically significant difference in the stem hardiness between the ploidy levels. The mean hardiness of the `PJM' florets was 211.18C and of the NSB florets 28.18C. Since in other studies `PJM' florets have been much more cold tolerant, it seems that the conditions in the phytotron did not allow full acclimation.