EFFICACY OF NOVEL WATER DISINFECTION TECHNIQUES IN HORTICULTURAL NUTRIENT RECYCLING.

Hydroponic systems used for growing potted ornamentals in greenhouses are commonly ebb-and-flow irrigation systems. The drainage water is usually recycled to save water and nutrients. To avoid the spread of pathogens in these closed irrigation systems, disinfection of the recycled water is standard practice. Growers can use slow sand filtration or UV-radiation techniques, but these methods are often either not sulted for specific problems or they require an excessively large investment. The objective of this study was to test less expensive but effective alternative disinfection systems. The efficacy of five disinfection systems against fungi and oomycetes was determined: Aqua-Hort (based on Cu-ions), Reciclean (performic acid), D1-OX Forte (CIO2), ECA (electrochemically activated water = anodic oxidation: hypochlorite and free radicals) and Newtec (also anodic oxidation). These five systems and a no-sterilization control were integrated in small closed ebb-and-flow circuits with nutrient solution reservoirs of 400 L each. Activity against Fusarium was excellent with ECA, good with Newtec and DI-OX Forte, moderate with high doses of Reciclean (250 ppm H2O2 and poor with the Aqua-Hort. There was no Pythium in the ECA and Newtec systems, while still so in the Aqua-Hort system, even at high doses (up to 7 ppm Cu++). Although the Reciclean (up to 100 ppm H2O2) and Aqua-Hort systems did not perform well against the pathogens, they did very well against algae; especially Reciclean was also useful against duckweed in water and liverwort on soil substrates. Concentrations of total Cl were elevated in water, substrate and plants after treatments with ECA and Newtec; other accumulations were Cu (Aqua-Hort), Na and SO4 (DI-OX Forte). However, only on a limited number of plant species these accumulations produced phytotoxic effects.

An application of serially balanced designs for the study of known taste samples with the α-ASTREE electronic tongue.

The α-ASTREE e-Tongue instrument uses seven sensors to characterize taste signals associated with a liquid sample. The instrument was used to study eight test preparations (comprised of a blank, four preparations corresponding to four known tastes and Sodium Topiramate in three concentrations known to have a bitter taste) and eight washes. Serially balanced residual effects designs were used to order the samples to estimate residual and main effects. The design provided for eight repeated measurements per test preparation. The experimental results suggested the following: (1) The seven sensors can be separated into three groups according to the ability to discriminate test preparations, and three of the sensors contributed little or no information. Further investigation suggested the lack of differentiability might be due to the age of the sensors. (2) The sensors discriminated known tastes from blank. The residual effect due to test preparations might appear after repeated usage. (3) Exploratory principal component analysis of the data indicated that nearly 90% of the total variability across the seven sensors could be explained by a single principal component. (4) The four standard taste preparations did not correspond to orthogonal dimensions in the principal component axes. (5) The three Sodium Topiramate test preparations could neither be associated with the corresponding known bitter taste sample nor could the three doses be shown to follow a quantitative dose-response relationship on the e-Tongue measurement scale. The practical interpretation of the results of the statistical analysis indicates only poor discriminative ability of the e-Tongue to distinguish clearly between increasing concentrations of a known bitter compound such as Sodium Topiramate. No apparent linear relationship could be discerned over increasing concentrations that would allow the quantification of bitterness.

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 ß-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 ß-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. References: (1) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul, MN, 2002. (2) P. W. Crous et al. Stud. Mycol. 55:213, 2006. (3) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997.

First Report of Cochliobolus sativus on Guzmania sp. in Belgium.

In 2001 and again in December 2005, an outbreak of leaf spots was observed on Guzmania sp. 'Gwendolyne' (Bromeliaceae) in a Belgian nursery. Typical disease symptoms were irregular spots with a grayish center and a narrow red-brown margin. Identification was based on morphological characteristics and molecular techniques. Isolations of diseased leaf tissues previously washed with sterile distilled water on potato dextrose agar (PDA) resulted in mycelial colonies after 7 to 8 days. Fungal mycelium grew at a linear rate of 30.4 mm per 24 h at 21°C in the dark. The pathogen produced aerial mycelium and sporulation was abundant. The color of the colonies on PDA was pale to dark brown and conidial characteristics similar to those of Cochliobolus sativus (anamorph Bipolaris sorokiniana) (1) were observed: brown ellipsoidal spores rounded at the top, 3 to 12 distoseptate, with average dimensions of 40 to 120 × 17 to 28 µm. The pathogen was also characterized with molecular tools. DNA was isolated from mycelium from a PDA plate. The ribosomal DNA region ITS1-5.8S-ITS2 was amplified and cloned. The ITS1 sequences (174 bp) of two independent clones were analyzed. The three highest similarity scores (E = 2e-71) obtained in BLAST were C. sativus (GenBank Accession Nos. AF158105 and AF071329) and B. sorokiniana strain BS11 (GenBank Accession No. AY372677). For these, pairwise alignments resulted in an identical score of 97.1% (169 identical bases, four indels, and one transversion). The new Genbank Accession No. of the ITS1 sequence is DQ 641269. To prove pathogenicity of the isolate, inoculations were done by spraying leaves of three young Guzmania sp. 'Gwendolyne' plants with a 20-ml spore suspension (106 spores/ml). Three plants were sprayed with sterile distilled water as controls. The plants were kept for 48 h under a humid chamber and subsequently at room temperature (20 to 25°C) on the laboratory bench. Three to four days after inoculation, leaf spots were observed and C. sativus (anamorph B. sorokiniana) was reisolated, completing Koch's postulates successfully. On the basis of symptoms, morphological characteristics, and pathogenicity tests, the pathogen was identified as C. sativus (anamorph B. sorokiniana). To our knowledge, this is the first record of C. sativus (anamorph B. sorokiniana) on Guzmania sp. in Belgium. References: (1) A. Sivanesan and P. Holliday. Description of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, England, UK, 1981.

First Report of White Rust Caused by Albugo tragopogonis on Sunflower in Belgium.

Sunflower (Helianthus annuus) is widely used for cut flowers and decoration in Belgium. A serious outbreak of what was suspected to be white rust on sunflower was observed in an East Flemish nursery near the city of Ghent in August 2004. This disease has previously been reported in Europe (southwest of France) (1) and other parts of the world with losses as much as 70 to 80% (Australia, North and South America, and Africa) (2,3). In the Flemish nursery, only single diseased plants (cv. Sunrich) were found. Blister-like pustules containing sporangia were observed on infected leaves. Initially the blisters were pale green to yellow on the abaxial surface and white on the adaxial surface of the leaves. As the disease progressed, white pustules that formed on the adaxial surface of the leaves slowly turned yellow, and the blisters on the abaxial surface became yellow to orange and necrotic in the center. Finally, the pustules coalesced and the leaves withered. Stem lesions were not observed. Short, cylindrical to spherical-cuboid sporangia, recovered from the pustules on the adaxial surface of leaves, measured between 17.5 and 22.5 µm, with an average of 20.2 µm. Sporangial dimensions were similar to those of Albugo tragopogonis (Pers.) S.F. Gray (1). Inoculations were done by spraying a suspension of 1 × 105 sporangia per ml prepared by scraping pustules from naturally infected leaves. Leaves on three 2-month-old healthy plants were sprayed with this inoculum and three plants sprayed with distilled water served as controls. The plants were kept for 48 h under a humid chamber and subsequently at room temperature (20 to 25°C) on the laboratory bench. Initial symptoms of white rust were observed 12 to 14 days after inoculation. On the basis of symptoms, morphological characteristics, and pathogenicity tests, the pathogen was identified as A. tragopogonis. To our knowledge, this is the first record of A. tragopogonis on H. annuus in Belgium. References: (1) K. G. Mukerji. Description of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, England, UK, 1976. (2) A. Pernaud and A. Perny, Phytoma 471:43, 1995. (3) P. S. van Wyk et al. Helia 22:83, 1999.

First Report of Leaf Spots on Laurus nobilis Caused by Phytophthora citricola in Belgium.

Laurus nobilis (laurel tree) is used as an herbal and ornamental tree in gardens in Belgium. During the summer of 2001, a serious outbreak of leaf spots was observed in some Belgian nurseries. Symptoms were large, irregular, brown leaf spots located primarily on the distal half of leaves and delimited by a black margin. As the disease progressed, the spots enlarged, coalesced, and finally led to leaf withering. The isolated fungus had morphological characteristics typical of Phytophthora citricola. On potato dextrose agar (PDA) it formed white, entire rosette colonies. Within 1 week, semipapillate sporangia were found abundantly in water at room temperature. They were mostly ovoid and highly variable in size (20 to 82 × 12 to 48 µm). The sporangial apex was broadly papillate. Oogonia were spherical (18 to 38 µm in diameter), and the antheridia were paragynous. Pathogenicity of the isolated fungus was confirmed by inoculating two visibly healthy L. nobilis plants. Five leaves per plant were wounded by a scalpel, subsequently inoculated with 5-mm-diameter mycelial plugs on PDA, and sealed with Parafilm. As a control, noncolonized agar plugs were placed on wounded leaves from a third L. nobilis plant. The inoculated plants and the control plant were kept for 1 day under a plastic cover (approximately 95% relative humidity) on the laboratory bench. Within 1 week, all inoculated leaves developed symptoms, whereas the control leaves remained symptomless. P. citricola was sucessfully re-isolated, satisfying Koch's postulates. The pathogen was also characterized using molecular tools. The ribosomal DNA regions, ITS1 and 5.8S rDNA-ITS2, were sequenced and highest similarity scores were obtained with corresponding Phytophthora citricola sequence regions (>99% identity for both sequences). The new GenBank Accession Nos. are AY525786 (ITS1) and AY525787 (5.8S rDNA-ITS2). On the basis of the symptoms, cultural and morphological characteristics, and positive results in pathogenicity and PCR tests, the isolate was considered to be P. citricola. To our knowledge, this is the first record of P. citricola on leaves of L. nobilis in Belgium.

First Report of Blight on Buxus spp. Caused by Cylindrocladium buxicola in Belgium.

In the fall of 2000, a new blight disease was observed on Buxus spp. in private gardens in Belgium. Since then, more and similar disease samples from other Belgian sites, nurseries, and several garden centers have been received, indicating that this disease is spreading. Similar observations have been made in the U.K. and France, where the disease is widespread and losses are sometimes dramatic (1). Diseased plants have dark brown-to-black leaf spots and streaky, black stem lesions which lead, in some cases, to complete defoliation. On some infected plants new leaves grew in defoliated areas, hiding the original blight symptoms. Infection was mainly observed on Buxus sempervirens cv. Suffruticosa, but B. sempervirens cv. Latifolia raculata, B. microphylla cv. Compacta, and B. microphylla var. japonica cv. Faulkner were also infected. In the U.K., infections have additionally been reported on varieties of B. sempervirens, B. sinica, and B. microphylla (1). On the basis of observed symptoms and comparison of the symptoms with descriptions by Henricot and Culham (2), we identified that this new form of Buxus blight in Belgium is caused by Cylindrocladium buxicola. Sporulating cultures on potato dextrose agar (PDA) had macroconidiophores with stipe extensions terminating in broadly ellipsoidal vesicles with pointed or papillate apices (6.5 to 11 µm in diameter) and a penicillate arrangement of fertile branches each terminating in two to five phialides. Phialides produced clusters of cylindrical conidia (42 to 68 × 4 to 6 µm) that were rounded at both ends and had a single septum. Pathogenicity of the isolate was demonstrated by inoculation of healthy stems and leaves of four 3-year-old plants of B. sempervirens cv. Suffruticosa. On each plant, agar pieces of 1-week-old cultures grown on PDA were placed on five stems and five leaves that had been wounded with a sterile scalpel, then sealed with Parafilm. As a control, five wounded stems and leaves from another B. sempervirens cv. Suffruticosa plant were inoculated with sterile agar plugs. Inoculated plants were incubated in humid chambers (approximately 95% relative humidity) on the laboratory bench. Two weeks after inoculation, no symptoms were visible on the control plant. The inoculated plants showed symptoms as previously described, and C. buxicola was successfully reisolated from diseased tissue completing Koch's postulates. To our knowledge, this is the first record of C. buxicola on Buxus spp. in Belgium. References: (1) B. Henricot et al. Plant Pathol. 49:805, 2000. (2) B. Henricot and A. Culham. Mycologia 94(6):980, 2002.

First Report of Plasmopara petroselini on Parsley in Belgium.

Plasmopara petroselini (Oomycetes) was identified on parsley (Petroselinum crispum subsp. crispum cv.Petra) (Apiaceae) in Belgium during the winters of 2001 and 2002. The fungus was present in numerous fields, especially on parsley grown in plastic tunnels. Losses were sometimes dramatic and similar to disease problems in France and Switzerland where 80 and 50 ha, respectively, were found infected (1). Initial symptoms consisted of white spots on the upper leaf surface. As the disease progressed, the spots enlarged, became angular, and turned yellow. At the location of leaf spots, white-to-grayish white mycelium developed on the lower surface of the leaves. Eventually the leaves and leaf stalks rotted. The pathogen was identified at the Centraalbureau voor Schimmelcultures (CBS) (Utrecht, the Netherlands) as the downy mildew organism P. petroselini (= P. umbelliferarum pro parte = P. nivea pro parte = P. crustosa), based on morphological characteristics. Sporangia were papillate, lemon-shaped, almost hyaline, and 9 to 20 µm long, and produced on tree-like sporangiophores (100 to 420 × 6 to 8.5 µm) that were monopodially branched at approximately right angles. The sporangiophores usually bear three sterigma (4 to 19 × 2 to 3 µm) that narrow toward the tip (2). Prophylactic actions are the primary method to prevent the disease. Fungicides based on propamocarb can be used as a curative control method. To our knowledge, this is the first report of P. petroselini on parsley in Belgium. References: (1) E. Béliard and J. Thibault. Phytoma 554:2, 2002. (2) M. Brandenburger. Page 451 in: Parastische Pilze an Gefässpflanzen in Europa. Fischer Verlag, Stuttgart, Germany, 1985.

Phytopthora ramorum in Belgium: 2002 survey results and research efforts.

Phytophthora ramorum is a new and aggressive Phytophthora species that causes leaf blight and dieback symptoms on Viburnum and Rhododendron plants in Europe. A variant of this fungus is responsible for Sudden Oak Death (SOD) in California and Oregon. In Europe, problems so far are mostly restricted to nursery plants of Rhododendron and Viburnum while in the US, the fungus has been isolated from over 20 host species and is responsible for massive killing of oak trees (mostly Quercus agrifolia and Lithocarpus densiflorus) in forest and park settings. The potential for infection of native tree species in Europe and the recent detection of the fungus in nurseries of several European countries has lead to the implementation of EU emergency phytosanitary measures. As a result, most European countries have conducted surveys and are doing research as part of risk assessment efforts. The first part of this paper focuses on the plant diagnoses of the 2002 survey of P. ramorum in Belgian nurseries. The data from the survey indicates P. ramorum is present in Belgium at similar rates as in the neighbouring countries, in an apparent random distribution. The second part of this paper describes research results relating to the in vitro effect of oomycete fungicides on P. ramorum, Rhododendron cultivar susceptibility, the determination of the leaf infection site, and pathogen survival. Some fungicides had excellent in vitro activity against P. ramorum and should be tested further on plants. Use of host resistance as a control strategy may be limited as little difference in cultivar sensitivity was observed. Infection studies showed that wounds and the lower sides of the leaves are most susceptible to infection. Once the pathogen gets inside, it can survive well on detached leaves, especially when they are kept cool and moist. These data can contribute to management decisions of P. ramorum at the level of nurseries as well as the government.

First Record of Leaf Spots on Prunus laurocerasus in Belgium Caused by Phytophthora cactorum and Peronospora sparsa.

In fall 2000 and 2001, large leaf spots were observed on Prunus laurocerasus. Two different plant pathogens proved to be the cause. Based on morphological characteristics they were identified as Peronospora sparsa (downy mildew) and Phytophthora cactorum. The P. cactorum isolate (CBS 110121) was identified at the Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands). Sporangia were papillate, ovoid, and deciduous, with a short pedicel. The isolate was homothallic. Chlamydospores were present and approximately 40 µm in diameter. Oogonia were 25 to 31 µm in diameter, and the antheridia were paragynous. Peronospora sparsa had been reported to infect Prunus laurocerasus in the United Kingdom (1). In Belgium, cv. Etna was very susceptible, but cvs. Rotundifolia and Marbled White were also infected. Rotundifolia was susceptible to P. cactorum. At first inspection, the two pathogens caused similar leaf symptoms: large, irregular, brown, necrotic spots on the tips, margins, and center of leaves. However, the undersides of leaves infected with Peronospora sparsa were covered with typical gray mycelium, which was absent on leaves infected with P. cactorum. P. cactorum caused concentric circles in the brown spots. Leaf spots caused by P. cactorum developed quickly in a moist chamber. Spots caused by Peronospora sparsa did not enlarge significantly on detached leaves, but in the field it caused serious losses within a few days. To prove the pathogenicity of P. cactorum, Koch's postulates were satisified on five Prunus laurocerasus Etna plants. The fungus was grown on corn meal agar for 1 week until sporangia formed. An agar plug was placed on five wounded leaves per plant and sealed with Parafilm. Inoculated plants were kept under a plastic cover for 1 day at 22°C, then the cover was removed, and the plants were kept at 20°C. Symptom development was visible after 3 days, and P. cactorum was successfully reisolated. This is the first record of P. cactorum and Peronospora sparsa leaf infection on Prunus laurocerasus in Belgium. Reference: (1) G. Hall et al. Plant Pathol. 41:224, 1992.

Pharmacokinetics and anti-trichomonal efficacy of a dimetridazole tablet and water-soluble powder in homing pigeons (Columba livia).

The anti-trichomonal efficacy and pharmacokinetics of dimetridazole were investigated in the homing pigeon (Columba livia). Dimetridazole was formulated for drinking water medication and as a prolonged-release tablet. To suppress a Trichomonas gallinae infection successfully, medicated drinking water containing dimetridazole (400 mg/L) had to be administered for at least 3 days. A two-day treatment with a dimetridazole tablet (20 mg/tablet) in fasted, as well as in fed, pigeons was shown to be ineffective. After intravenous administration of 20 mg dimetridazole, the drug plasma concentration-time profile fitted a one-compartment open model with a mean half-life of 3.9 h. The absolute bioavailability of the tablet in fasted pigeons was 83.8%. The bioavailability of the tablet administered with food was reduced by 20%. Dimetridazole was rapidly metabolised to (1-methyl-5-nitroimidazol-2-yl) methanol.