Back to the Roots: Agrobacterium-Specific Phages Show Potential to Disinfect Nutrient Solution from Hydroponic Greenhouses.

Agrobacterium biovar 1 is a soilborne plant pathogen with the ability to colonize the irrigation system of greenhouses, causing hairy root disease (HRD). Currently, management focuses on using hydrogen peroxide to disinfect the nutrient solution, but due to the emergence of resistant strains, its efficacy and sustainability are questioned. Using a relevant collection of pathogenic Agrobacterium biovar 1 strains, OLIVR1 to 6, six phages specific to this pathogen and belonging to three different genera were isolated from Agrobacterium biovar 1-infected greenhouses. All phages were named OLIVR, referring to their location of isolation, Onze-Lieve-Vrouwe-Waver, and were characterized by whole-genome analysis, confirming their strictly lytic lifestyle. They remained stable under greenhouse-relevant conditions. To assess the efficacy of the phages, their ability to disinfect greenhouse nutrient solution inoculated with agrobacteria was tested. Each of the phages infected their host, but their ability to decrease the bacterial concentration differed. For instance, OLIVR1 reduced the bacterial concentration with 4 log units without phage resistance emerging. While OLIVR4 and OLIVR5 were also infectious in nutrient solution, they did not always decrease the bacterial load below the limit of detection, and phage resistance emerged. Finally, the mutations causing phage resistance by receptor modification were identified. For OLIVR4-resistant Agrobacterium isolates, but not for OLIVR5-resistant isolates, motility decreased. Together, these data show the potential of some of these phages as disinfectant of nutrient solution, and they might be a valuable tool to tackle HRD. IMPORTANCE Hairy root disease, caused by rhizogenic Agrobacterium biovar 1 is a rapidly emerging bacterial disease worldwide. It affects tomatoes, cucumbers, eggplant, and bell pepper, causing high yield losses in hydroponic greenhouses. Recent findings suggest that the current management practices, mainly focusing on UV-C and hydrogen peroxide to disinfect contaminated water, have a questionable efficacy. Hence, we investigate the potential of phages as a biological means of preventing this disease. Using a diverse collection of Agrobacterium biovar 1, we isolated three different phage species that together infect 75% of the collection. Since these phages are strictly lytic, while remaining both stable and infectious under greenhouse-relevant conditions, they might be suitable candidates for biological control.

First Record of Bacterial Blight Caused by Pseudomonas syringae pv. syringae on Pyracantha coccinea and an Amelanchier sp. in Bulgaria.

During the spring and summer months of 2004 and 2005, sporadic damage on individual shrubs of Pyracantha coccinea and an Amelanchier sp. were observed at two locations in the region of Plovdiv, Bulgaria. Symptoms initially were expressed as blossom blight and subsequently expanded to the shoots and branches, forming cankers on the supplying wood. In both years, a fluorescent gram-negative bacterium was isolated from diseased tissues onto King's B medium. The bacterial strains were levan positive and oxidase and arginine dihydrolase negative. They were able to induce a typical hypersensitive response on tobacco plants (cv. Samsun), but failed to rot potato slices. Pathogenicity of the strains was confirmed by puncture-inoculating detached shoots from both hosts and immature cherry and pear fruits with a bacterial suspension (108 CFU/ml, 50 µl per wound, and 3 replicates). Controls were punctured with sterile water. The inoculated plant material was maintained at room temperature (22 to 25°C) in plastic pots and covered with polyethylene bags for the first 48 h after inoculation. The inoculated and control subjects were kept under the same conditions as before inoculation. Except for the controls, slowly expanding but well defined necrotic lesions around the inoculation points were observed within the next 5 to 7 days. Bacteria reisolated from symptomatic tissue were identical to the initial cultures. On the basis of the symptoms and results from all laboratory tests, the bacterium was considered to be Pseudomonas syringae pv. syringae (1). PCR amplification of the 752-bp syrB fragment (2) confirmed the identification. To our knowledge, this is the first occurrence of P. syringae pv. syringae on Pyracantha coccinea and an Amelanchier sp. in Bulgaria, and most probably, this pathogen will play a more significant role within the rosaceous group because of a rising number of the cultivated ornamental species. References: (1) N. W. Schaad et al., eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001. (2) K. N. Sorensen et al. Appl. Environ. Microbiol. 64:226, 1998.

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 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 Fire Blight Caused by Erwinia amylovora on Pyracantha coccinea in Bulgaria.

During late May and June of 2003, a fire blight epidemic occurred in southcentral Bulgaria on earlier reported hosts such as apple, pear, quince, and hawthorn (1). A new host was found when fire blight was also observed in mid-June on Pyracantha coccinea grown in the Plovdiv Region. Symptoms were necrotic flowers, shoots, petioles, and the presence of sticky ooze droplets mainly on the shoots. Isolations made from blighted Pyracantha coccinea flowers and shoots onto King's medium B (2 days at 25 to 26°C) yielded whitish, glistening, round bacterial colonies. Infiltration of the suspensions of three of the isolates into tobacco leaves resulted in a typical hypersensitive reaction. When Pyracantha coccinea and Cotoneaster sp. shoots were inoculated with these three isolates, typical fire blight symptoms were obtained. The pathogen was reisolated 2 weeks after inoculation from necrotic tissues (15 to 20 mm above and below the inoculation site), thereby fulfilling the Koch's postulates. No symptoms and bacteria were found within any of the shoots from the same plant species injected with sterile water. The identity of the isolates was also determined by conducting nested polymerase chain reaction (PCR) (2) and target-specific PCR (23S rDNA) (3). On the basis of the symptoms, cultural characteristics, and positive results in pathogenicity and PCR tests, the isolates were considered to be Erwinia amylovora. To our knowledge, this is the first report of fire blight on Pyracantha coccinea in Bulgaria. References: (1) S. G. Bobev et al. Plant Dis. 82:1283, 1998. (2) P. Llop et al. Appl. Environ. Microbiol. 66:2071, 2000. (3) M. Maes et al. Plant Pathol. 45:1139, 1996.

First Report of Phytophthora cactorum on American Ginseng (Panax quinquefolius) in Bulgaria.

American ginseng (Panax quinquefolius) is a recently introduced crop in Bulgaria. In autumn 2001, several 2-year-old plants from Stara Zagora County exhibited symptoms of wilting and dying. Laboratory analysis also revealed some browning of the ginseng root surface and discoloration of the vascular tissues. During later stages of the disease, roots became soft, rubbery, and disintegrated. After storage in a humid chamber for 3 to 5 days, roots were covered with a white, cottony mycelium. Following the transfer onto potato dextrose agar, this fungus formed rounded colonies of white, aerial mycelium. Pathogenicity of the isolate was demonstrated by inoculation of roots that were surface-disinfected with alcohol (70%) for 30 s and rinsed with sterile water. Roots were wounded with a scalpel, and agar pieces from a 1-week-old culture were placed under the cortical tissue. Five inoculated root pieces were kept in a humid chamber at 24 to 25°C, and the pathogen was reisolated subsequently from necrotic lesions that developed from wounds. No symptoms were found in the five wounded but noninoculated control roots. The pathogen was reisolated from the diseased tissue to fulfill Koch's postulates. Microscopic examination showed that the pathogen had an aseptate mycelium (mean diameter of 5.3 µm), did not form hyphal swellings or chlamydospores, and had simple sympodial branching of the sporangiophores. Sporangia had a caducous nature with a pedicel length of 4.7 µm (1.7 to 6.7 µm). Sporangia were ovoid to obpyriform in shape, papillate, and nonproliferating measuring 30.6 (26.6 to 40.0) µm × 24.3 (23.3 to 30.0) µm. The length/width ratio varied between 1.25 and 1.3. The fungus was homothallic and produced paragynous antheridia and spherical oogonia with a diameter of 30.6 µm (26.6 to 33.3 µm) on V8 agar and in petri solution. Oospores were aplerotic and spherical (25 to 30 µm in diameter). Based on symptoms and pathogen characteristics (2), the disease was identified as Phytophthora root rot caused by Phytophthora cactorum. Additionally, the identity of the isolate was verified by sequence determination of the ribosomal internal transcribed spacer I region and alignment to the GenBank-EMBL DNA database (1), which revealed 100% sequence similarity with P. cactorum. To our knowledge, this is the first report of P. cactorum on American ginseng in Bulgaria. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389,1997. (2) D. C. Erwin and O. K. Ribeiro. Morphology and identification of Phytophthora species. Pages 96-125 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996.

Experiences and perspectives for the use of a Paenibacillus strain as a plant protectant.

A study on the microbial ecology in an active slow sand filter, used for disinfecting the circulating plant nutrient solutions, showed that spore-forming plant-associated bacteria belonging to the Bacillus-Paenibacillus complex are well adapted for transmission in the solutions and passage through the filter. Therefore, strains from this bacterial group were suitable candidates for biological control in irrigated and closed plant growth systems. The spore-forming Paenibacillus polymyxa strain PpDGB was selected in in vitro tests as a potent pathogen-antagonist and was tested as a prophylactic protection agent in the plant rhizosphere, especially for cultures stages that are highly susceptible to stress and disease. Plant cuttings, in vitro plants and seeds of different plant types were bacterized and planted in their typical disease-conducive environment where nutrient solutions or water irrigation was applied and further plant development was monitored. Observed plant parameters were plant survival, weight, chlorophyll concentration in the leaf mesophyl, root health and root hair formation. The PpDGB treatment initially induced stress in the plants, which was observed as a transient stop in plant transpiration. This effect caused some necrosis in the most stress-sensitive in vitro plant species. In the other plants this stress period was followed by a significant enhancement in plant growth. In case of seed treatment, more seeds germinated and seedling growth was faster. In the tested formulation, PpDGB enhanced growth but not disease resistance, probably due to simultaneous activation of the residual plant pathogens. Therefore variant formulations have to be tested. The influence of PpDGB on the composition of the bacterial communities in the rhizosphere was assessed by DGGE profiling. In soilless plant cultures, PpDGB-driven profile changes could be observed from the 5th day after the initial treatment. P. polymyxa bacteria were shown to be widely present in association with plants and specific PpDGB detection in plant and rhizosphere was only possible with newly developed strain-specific PCR primers based on Nif H gene sequences. Quantitative PCR based on SYBR Green fluorescence enabled detection of low PpDGB concentrations in the plant rhizosphere.