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.

Phylogenetic Assignment of Ralstonia pseudosolanacearum (Ralstonia solanacearum Phylotype I) Isolated from Rosa spp.

During the last two years, greenhouse cultivation of rose (Rosa spp.) in the Netherlands has been challenged by an uncommon bacterial disease. Affected plants suffered from chlorosis, stunting, wilting, and necrosis. The bacterial isolates obtained from the different Rosa spp. cultivars were all identified as phylotype I, sequevar 33 from the 'Ralstonia solanacearum species complex' (RSSC), actually reclassified as Ralstonia pseudosolanacearum. The work in this paper considers the genetic diversity and the phylogenetic position of 129 R. pseudosolanacearum isolates from the outbreak. This was assessed by AFLP based on four different primer combinations and MLP using partial sequences of the egl, mutS, and fliC genes. The AFLP revealed identical profiles for all the isolates, irrespective of their association with Rosa sp. propagating material, Rosa spp. plants for cut flowers, or water used in the different greenhouse cultivations. These AFLP profiles were unique and diverged from profiles of all other reference isolates in the RSSC included. Furthermore, MLP on egl, fliC, and mutS gene sequences clearly demonstrated that all R. pseudosolanacearum isolates clustered in phylotype I, as a distinct monophyletic group. Interestingly, this monophyletic group also included phylotype I strain Rs-09-161 from eggplant (Solanum melongena), isolated in 2009 in India. AFLP and MLP were both efficient in revealing the genetic divergence from the RSSC isolates included. The phylogenetic tree constructed from the AFLP profiles was, in general, in agreement with the one obtained from MLP. Both phylogenetic trees displayed a similar clustering, supported by high posterior probabilities. Both methodologies clearly demonstrated that the R. pseudosolanacearum isolates from Rosa spp. grouped in a monophyletic group inside phylotype I, with a particular correspondence to a strain present in India, as revealed in MLP.

First Report of Dickeya dianthicola Causing Blackleg on Potato (Solanum tuberosum) in Bulgaria.

Potato (Solanum tuberosum) is an important and widespread crop in Bulgaria. A new disease was observed on a single potato plot (Plovdiv region) without a history of potato cultivation in the spring of 2011. Initially, single lower leaves wilted on recently emerged plants (approx. 15% incidence) with subsequent desiccation of the leaf margins. The wilting progressed over time and eventually the whole stem became desiccated. A blackleg-like necrosis was noticed at the stem base when symptomatic plants were uprooted. Most diseased stems remained green above ground but pith tissue was heavily macerated and some of the stems became hollow as the pith dried out. Mother tubers were partially or entirely macerated. In most cases, the decay was initiated from the stolon end. Bacterial strains were obtained from symptomatic stems and tubers by dilution plating on King's B medium. The strains produced indigoidin pigment and induced a hypersensitive response 24 h after infiltration into tobacco and Sedum hybridum leaves (2). The strains were identified as Dickeya spp. by the production of the PCR amplicon of the pectate lyase ADE gene cluster (3) and of the pectate lyase I gene (4). The partial sequence of the fliC PCR amplicon (1) of strain SB2589 (GenBank Accession No. KF442436) displayed 100% homology with four whole genome shotgun sequences of Dickeya dianthicola in GenBank. Pectinolytic activity was demonstrated by inoculation of surface disinfested potato tubers of cv. Kondor. Conical core tissue was removed at the apical end and 100 µl bacterial suspension (107 CFU in sterile 10 mM phosphate buffer) was deposited in the cavity. The cap was reattached to the tuber and immobilized by Parafilm. Positive control tubers were inoculated with D. dianthicola reference strain GBBC 2039 (LMG 25864) and negative control tubers were inoculated with sterile 10 mM phosphate buffer. All tubers were incubated for 48 h at 28°C under micro-aerobic conditions reducing the air pressure to 90 mb in a vacuum incubator. The D. dianthicola reference strain and Bulgarian strains produced maceration of tuber tissue. Maceration was not observed in the negative control tubers. Potato plants cv. Kondor were grown from minitubers in sandy soil in plastic nursery containers. The plants were inoculated by root drenching (one application of cell suspension at 109 CFU/liter) when the stems were 15 to 20 cm high (tuber initiation stage). Plants were incubated at 25 to 28°C with regular watering. Wilting symptoms developed within 10 days of inoculation, followed by necrosis of the pith. Strains obtained from the inoculated stems were confirmed as D. dianthicola as described above. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the disease was caused by D. dianthicola and to our knowledge this is the first report of the pathogen on potato in Bulgaria. Furthermore, this incident warrants further surveys of pectinolytic bacteria causing blackleg-like symptoms in potato crops in Bulgaria. References: (1) S. Diallo et al. Eur. J. Plant Pathol. 125:349, 2009. (2) Y-.A. Lee and C-.P. Yu. J. Microbiol. Methods 64:200, 2006. (3) A. Nassar et al. Appl. Environ. Microbiol. 62:2228, 1996. (4) J. Van Vaerenbergh et al. PLoS ONE 7(5):e35738, 2012.

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 Fire Blight on Pyrus elaeagrifolia and Amelanchier sp. in Bulgaria.

In 2005, a fire blight epidemic occurred for the second time within the last 3 years, and severe damages were observed on pome fruits trees in many regions of Bulgaria. For the first time, we found fire blight on Pyrus elaeagrifolia and Amelanchier sp. grown in a park area (Plovdiv Region), providing evidence of continuing spread of the pathogen in Bulgaria. The symptoms on P. elaeagrifolia were necrotic, immature fruitlets and progressive necrosis toward the adjacent branches, thus forming cankers and leading to death of the plant above the canker. Many Amelanchier sp. shrubs had severely blighted flowers, fruitlets, shoots, and branches and dried, amber ooze droplets on the shoots. All the isolations made from blighted hosts' shoots and cankers on King's medium B (2 to 3 days, 26 to 27°C) yielded whitish, glistening, round bacterial colonies. Infiltration of the suspensions of randomized isolates from both hosts into tobacco leaves resulted in a typical hypersensitive reaction. Subsequently, some strains showed a typical ooze production on immature pear fruits (cv. Conference) and were also successfully reisolated from artificially inoculated quince shoots (1.2 × 109 CFU, cv. Portugalska, three replicates), where typical fire blight symptoms were observed, thereby fulfilling Koch's postulates. No symptoms or bacteria were found within any of the shoots from the same plant species injected with sterile water. The identity of the isolates was confirmed as Erwinia amylovora by an antibody-based slide agglutination test (Neogen_Express; Neogen Europe, Ltd., UK) and PCR test with primers derived from the ams region (1). On the basis of the symptoms, cultural characteristics, and positive results in pathogenicity, serological, and PCR tests, the isolates were considered to be E. amylovora. To our knowledge, this is the first report of fire blight on P. elaeagrifolia and Amelanchier sp. in Bulgaria. Reference: (1) S. Bereswill et al. Appl. Environ. Microbiol. 61:2636, 1995.

Pseudomonads associated with midrib rot and soft rot of butterhead lettuce and endive.

During the past ten years, bacterial soft rot and midrib rot of glasshouse-grown butterhead lettuce (Lactuca sativa L. var. capitata) and field-grown endive (Cichorium endivia L.) has become increasingly common in the region of Flanders, Belgium. Severe losses and reduced market quality caused by bacterial rot represent an important economical threat for the production sector. Symptoms of midrib rot are a brownish rot along the midrib of one or more inner leaves, often accompanied by soft rot of the leaf blade. Twenty-five symptomatic lettuce and endive samples were collected from commercial growers at different locations in Flanders. Isolations of dominant bacterial colony types on dilution plates from macerated diseased tissue extracts yielded 282 isolates. All isolates were characterized by colony morphology and fluorescence on pseudomonas agar F medium, oxidase reaction, and soft rot ability on detached chicory leaves. Whole-cell fatty acid methyl esters profile analyses identified the majority of isolates (85%) as belonging to the Gammaproteobacteria, which included members of the family Enterobacteriaceae (14%) and of the genera Pseudomonas (73%), Stenotrophomonas (9%), and Acinetobacter (3%). Predominant bacteria were a diverse group of fluorescent Pseudomonas species. They were further differentiated based on the non-host hypersensitive reaction on tobacco and the ability to rot potato slices into 4 phenotypic groups: HR-/P- (57 isolates), HR-/P+ (54 isolates), HR+/P (16 isolates) and HR+/P+ (35 isolates). Artificial inoculation of suspensions of HR-, pectolytic fluorescent pseudomonads in the leaf midrib of lettuce plants produced various symptoms of soft rot, but they did not readily cause symptoms upon spray inoculation. Fluorescent pseudomonads with phenotype HR+ were consistently isolated from typical dark midrib rot symptoms, and selected isolates reproduced the typical midrib rot symptoms when spray-inoculated onto healthy lettuce plants.

A neural network for recognizing movement patterns during repetitive self-paced movements of the fingers in opposition to the thumb.

A data glove is a typical input device to control a virtual environment. At the same time it measures movements of wrist and fingers. The purposes of this investigation were to assess the ability of BrainMaker, a neural network, to recognize movement patterns during an opposition task that consisted of repetitive self-paced movements of the fingers in opposition to the thumb. The neural network contained 56 inputs, 3 hidden layers of 20 neurons, and one output. The 5th glove '95 (5DT), a commercial glove especially designed for virtual reality games, was used for finger motion capture. The training of the neural network was successful for recognizing the thumb, the index finger and the ring finger movements during the repetitive self-paced movements and neural network performed well during testing.

[Positive Romberg test and the probability of falls in the aged]. / Positieve Romberg-test en het voorspellen van vallen bij bejaarden.

Very little objective data are available on the subject of falling in the elderly. Yet there is an urgent need for simple tests to help identify those patients at risk. The predictive value of the Romberg-test was investigated with a group of 199 inhabitants of four homes for the elderly (76 had fallen three times in a period of six months and 123 never fell in a period of one year). Beside the age factor a positive Romberg-test meant an increased chance of falling. Elderly patients who fell but showed up as negative on the Romberg-test showed a marked tendency to stumble compared with the control group. The Romberg-test is useful in identifying certain specific causes in falling but it is unsuitable for use within a preventative strategy.

[Electromyography study of the anterior tibial muscle and accidental falls in the elderly]. / Die elektromyographische Untersuchung des Musculus tibialis anterior und das Fallen alter Menschen.

This article investigates if surface electromyography of the tibialis anterior muscle during rest, maximal contraction and balance stressing pick out elderly people at risk for falling. With a same view postural responses underlying balance control were recorded as well. Four groups were compared: an experimental group composed of nursing home residents (n = 15) with three or more unexplained falls in the prior year, a nonfalling control group of nursing home residents (n = 15) of equivalent age, a group of subjects (n = 15) in middle age and finally a group of young participants (n = 15). EMG activity during maximal contraction of the tibialis anterior muscle was significantly lower in the falling group and the postural responses during balance testing were inappropriate.