First Report of Agrobacterium rubi and A. rhizogenes Causing Crown and Root Gall and Hairy Root on Blueberry in Argentina

From 2006 to 2009, crown gall and hairy root symptoms were observed on blueberry (Vaccinium corymbosum cvs. O'Neil, Millennia, and Misty) plants from six nurseries in Tucumán, Concordia, Pilar, Morón, and Baradero, Argentina. Bacteria were isolated from galls of all three cultivars and from hairy roots of Millenia and O'Neil onto D1 and D1M agar media at 27°C. Typical Agrobacterium colonies developed in 5 days (2). Seven bacterial strains (five from galls and two from hairy roots) were studied further. All were gram negative, aerobic, and catalase positive with rod-shaped cells that synthesized β–galactosidase and metabolized D-glucose, D-arabinose, n-acetyl-glucosamine, maltose, mannitol, and malonate. Strains were negative for lysine decarboxylase, H2S production, indole, and 3-ketolactose production. While gall strains were urease positive and citrate variable (mostly positive), hairy root strains were urease negative, citrate positive, had poly-β-hydroxybutyrate inclusion granules, and clarified acid on potato dextrose agar containing 0.5% CaCO3 (2). Agrobacterium tumefaciens ATCC 15955 and LBA 958 were included as controls. PCR with virA/C primers amplified a 338-bp product corresponding to the virD2 operon and confirmed that the strains harbored a pathogenic plasmid (1). Bacterial strains were assigned to biovars with a multiplex PCR assay targeting 23S rRNA sequences (3). Two strains produced PCR amplicons typical of A. rhizogenes bv. 2. The other five strains produced PCR amplicons typical of A. rubi, which were insensitive to agrocin in a bioassay with A. radiobacter strain K1026. Identity was confirmed by sequencing the 16S rDNA of strains F 266 (GenBank No. GU580894) and F 289 (No. GU580895), which had 99% homology to 16sRNA sequences of A. rubi ICMP 11833 (AY626395.1) and A. rhizogenes ATCC 11325 (AY945955.1), respectively. Pathogenicity of all seven strains was tested on V. corymbosum cv. Misty, Bryophyllum daigremontiana, tobacco cv. Xanthi, tomato cv. Presto, and pepper cv. California Wonder. Plants were inoculated by a needle stabbed into the stems with the appropriate cell suspension (108 CFU/ml) of each strain or with sterile distilled water (control treatment). Two plants of each species were tested per strain. Plants were grown for at least 45 days at 23 ± 3°C and symptoms were recorded. Inoculations with the five strains isolated from galls caused development of spherical, white to flesh-colored, rough, spongy wart-like galls at the inoculation sites. Root strains induced root proliferation on all inoculated plants as well as in a carrot disk bioassay (4). On blueberry plants, galls were dark brown to black, rough, and woody 6 months after inoculation. No lesions were observed on control plants. Bacteria were reisolated from symptomatic tissues of inoculated plants. Enterobacterial repetitive intergeneric consensus-PCR confirmed that the DNA fingerprints of the reisolated strains were identical to those of the original strains. To our knowledge, this is the first report of A. rubi and A. rhizogenes causing hairy root and crown gall on blueberry in Argentina. References: (1) J. H. Haas et. al. Appl. Environ. Microbiol. 61:2879,1995. (2) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) J. Pulawska et al. Syst. Appl. Microbiol. 29:470, 2006. (4) M. H. Ryder et al. Plant Physiol. 77:215, 1985.

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First Report of Agrobacterium vitis as Causal Agent of Crown Gall Disease of Grapevine in Tunisia

Plant Disease ◽

10.1094/pdis-12-12-1125-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 836-836 ◽

Cited By ~ 1

Author(s):

S. Chebil ◽

R. Fersi ◽

S. Chenenaoui ◽

E. Abdellatif ◽

G. Durante ◽

...

Keyword(s):

Multiplex Pcr ◽

Crown Gall ◽

Causal Agent ◽

Control Treatment ◽

Vitis Vinifera L ◽

Agrobacterium Vitis ◽

First Report ◽

Crown Gall Disease ◽

Multiplex Pcr Assay ◽

Ti Plasmids

Since October 2011, a serious outbreak of crown gall disease was observed on 1- and 2-year-old grapevines (Vitis vinifera L.) cv. Superior Seedless in several vineyards located in the region of Regueb in the center of Tunisia. Fifty isolates of Agrobacterium were isolated on a tartrate medium from galls of affected plants. To prepare template DNA, cell suspensions were lysed in 0.25% sodium-azide (NaN3) buffer prepared in 1% Triton X-100 by heating the samples at 95°C for 10 to 15 min (1). The strains were differentiated using a multiplex PCR assay with a combination of VIRFF1/VIRFR2 and VIRD2S4F716/VIRD2S4R1036 primers (2), which detect regions of virF and virD2 genes, respectively, in A. vitis strains carrying octopine or nopaline Ti plasmids and A. vitis vitopine strains. In order to differentiate A. vitis strains from A. tumefaciens strains, PGF/PGR (4), a polygalacturonase specific primer set, was added to the mixture in multiplex PCR. The isolates segregated into three main groups. The first group carries octopine type Ti plasmids, the second carries vitopine type Ti plasmids, and the third group carries both octopine and vitopine type Ti plasmids. The polygalacturonase gene sequence from 10 isolates showed 94 to 97% identity to the sequences of A. vitis previously deposited in the NCBI GenBank database (Accession No. CP000633.1gb). The biochemical test results corresponded to the results of genetic analysis. The ability to aerobically convert lactose to 3-ketolactose was tested by spotting bacteria onto medium containing lactose and flooding plates with a layer of Benedict's reagents after incubation at 28°C for 48 h. Acid production from glucose was tested by spotting bacterial strains onto potato dextrose agar (PDA) medium supplemented with CaCO3. Alkali production from L-tartrate was tested by streaking bacteria on AB minimal medium supplemented with L-tartrate and growth in salt medium was tested by streaking on nutrient broth supplemented with 2% NaCl. All isolates except one were negative in 3-ketolactose. They were negative in acid clearing on PDA-CaCO3, grew in 2% NaCl, and produced alkali from tartarate. Pathogenicity of all 50 strains was tested on 1-month-old tomato plants (Lycopersicum esculentum cv. Riograndi). Plants were inoculated on the stem by pricking one to three times through a drop of inoculum (108 CFU/ml) at three inoculation sites. Sterile distilled water was used as control treatment. Plants were grown for 4 weeks at 23 ± 3°C and symptoms were recorded. Typical tumors developed at the inoculation sites and no symptoms were observed on the control plants. In Tunisia, crown gall disease was observed only on stone fruit trees and only A. tumefaciens Biovar 1 have been reported and assigned to four genomic species G4, G6 G7, and G8 basically on the recA sequencing (3). To our knowledge, this is the first report of A. vitis determined as the causal agent of grapevine crown gall in Tunisia. References: (1) A. Abolmaaty et al. Microbios 101:181, 2000. (2) F. Bini et al. Vitis 47:181, 2008. (3) D. Costechareyre et al. Microb. Ecol. 60:862, 2010. (4) E. Szegedi and S. Bottka. Vitis 41:37, 2002.

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First Report of Blueberry Reddening Disease in Serbia Associated with 16SrXII-A (Stolbur) Phytoplasma

Plant Disease ◽

10.1094/pdis-05-13-0521-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1653-1653 ◽

Cited By ~ 3

Author(s):

M. Starović ◽

S. Kojic ◽

S. T. Kuzmanovic ◽

S. D. Stojanovic ◽

S. Pavlovic ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Nested Pcr ◽

Vaccinium Corymbosum ◽

23S Rrna ◽

Rrna Gene ◽

Restriction Enzyme Digestion ◽

First Report ◽

Stolbur Phytoplasma ◽

The 16S Rrna Gene

Blueberries (Vaccinium corymbosum) are among the healthiest fruits due to their high antioxidant content. The total growing area of blueberries in Serbia ranges from 80 to 90 ha. A phytoplasma-like disease was observed for the first time during July 2009 in three blueberry cultivars (Bluecrop, Duke, and Spartan) grown in central Serbia, locality Kopljare (44°20′10.9″ N, 20°38′39.3″ E). Symptoms of yellowing and reddening were observed on the upper leaves and proliferating shoots, similar to those already described on blueberries (4). There was uneven ripening of the fruits on affected plants. Incidence of affected plants within a single field was estimated to be greater than 20% in 2009 and 50% in 2010. Blueberry leaves, together with petioles, were collected during two seasons, 2009 and 2010, and six samples from diseased plants and one from symptomless plants from each cultivar, resulting in 42 samples in total. For phytoplasma detection, total DNA was extracted from the veins of symptomatic and asymptomatic leaves of V. corymbosum using the protocol of Angelini et al. (1). Universal oligonucleotide primers P1/P7 were used to amplify a 1.8-kb DNA fragment containing the 16S rRNA gene, the 16S-23S spacer region, and the 5′ end of the 23S rRNA gene. Subsequently, a 1.2-kb fragment of the 16S rRNA gene was amplified by nested PCR with the R16F2n/R16R2 primers. Reactions were performed in a volume of 50 μl using Dream Taq Green master mix (Thermo Scientific, Lithuania). PCR reaction conditions were as reported (3), except for R16F2n/R2 primers set (annealing for 30 s at 58°C). PCR products were obtained only from the DNA of symptomatic plants. Fragments of 1.2 kb were further characterized by the PCR-RFLP analysis, using AluI, HpaII, HhaI, and Tru1I restriction enzymes (Thermo Scientific, Lithuania), as recommended by the manufacturer. The products of restriction enzyme digestion were separated by electrophoresis on 2.5% agarose gel. All R16F2n/R2 amplicons showed identical RFLP patterns corresponding to the profile of the Stolbur phytoplasma (subgroup 16SrXII-A). The results were confirmed by sequencing the nested PCR product from the representative strain Br1. The sequence was deposited in NCBI GenBank database under accession number KC960486. Phylogenetic analysis showed maximal similarities with SH1 isolate from Vitis vinifera, Jordan (KC835139.1), Bushehr (Iran) eggplant big bud phytoplasma (JX483703.1), BA strain isolated from insect in Italy (JQ868436.1), and also with several plants from Serbia: Arnica montana L. (JX891383.1), corn (JQ730750.1), Hypericum perforatum (JQ033928.1), tobacco (JQ730740.1), etc. In conclusion, our results demonstrate that leaf discoloration of V. corymbosum was associated with a phytoplasma belonging to the 16SrXII-A subgroup. The wild European blueberry (Vaccinium myrtillus L.) is already detected as a host plant of 16SrIII-F phytoplasma in Germany, North America, and Lithuania (4). The main vector of the Stolbur phytoplasma, Hyalesthes obsoletus Signoret, was already detected in Serbia (2). The first report of Stolbur phytoplasma occurrence on blueberry in Serbia is significant for the management of the pathogen spreading in blueberry fields. Since the cultivation of blueberry has a great economic potential in the region, it is important to identify emerging disease concerns in order to ensure sustainable production. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) J. Jović et al. Phytopathology 99:1053, 2009. (3) S. Pavlovic et al. J. Med. Plants Res. 6:906, 2012. (4) D. Valiunas et al. J. Plant Pathol. 86:135, 2004.

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First Report of Fire Blight Caused by Erwinia amylovora on Meadowsweet (Spirea prunifolia) in Turkey

Plant Disease ◽

10.1094/pdis-03-13-0220-pdn ◽

2014 ◽

Vol 98 (1) ◽

pp. 153-153 ◽

Cited By ~ 2

Author(s):

K. K. Bastas ◽

F. Sahin

Keyword(s):

Pathogenic Bacteria ◽

Fire Blight ◽

Erwinia Amylovora ◽

Natural Infection ◽

Hypodermic Needle ◽

Identification System ◽

Bacterial Strains ◽

First Report ◽

Molecular Tests ◽

Initial Symptoms

Fire blight, caused by Erwinia amylovora (Burr.) Winslow et al., affects plants in the Rosaceae family, which includes trees and shrubs in orchards, nurseries, and landscape plantations. During the springs and summers of 2008 and 2010, dying branches, necrotic leaves attached to shoots, and blighted twigs of meadowsweet (Spirea prunifolia) were observed at three different locations of landscape areas in Konya Province, Turkey. Disease incidence was approximately 1% on the plants during the surveys. Initial symptoms of reddish to brownish streaks on the shoots of infected plants were observed in spring. Nine representative bacterial strains were isolated from the lesions on shoots of seven meadowsweet plants on nutrient sucrose agar (NSA) medium and identified as E. amylovora on basis of biochemical, physiological (2,3) and molecular tests (1). Bacteria were gram-negative, rod shaped, aerobic, fermentative, yellow-orange on Miller and Scroth medium (2), positive for levan formation and acetoin production, did not grow at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, acid production from lactose, and inositol. All strains were hypersensitive response-positive on tobacco (Nicotiana tabacum var. White Burley) plants. All strains were identified as E. amylovora using the species-specific primers set, A/B (1), by PCR assay, and by fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE) with similarity indices ranging from of 79 to 99%. Pathogenicity was tested by injecting of petioles and actively growing three shoot tips of 2-year-old S. prunifolia seedlings cv. number 29 using a 0.46 mm-diameter hypodermic needle with bacterial suspensions containing 108 CFU mL–1 in sterile distilled water (SDW) Plants were inoculated with each of the nine bacterial strains and two references strains, Ea29 and NCPPB 2791 (Selcuk University, Department of Plant Protection, Konya, Turkey). Symptoms resembling those associated with natural infection appeared on the inoculated plants 7 days after inoculation. Plants inoculated with SDW served as a negative control treatment, and no symptoms were observed on these plants. All tests were repeated three times with the same results. Bacterial re-isolations were attempted from the control plants as well as shoots and leaves inoculated with the two reference strains and the nine bacteria identified as E. amylovora. Bacteria isolated from inoculated plants were identified as E. amylovora using the biochemical, physiological, and molecular tests described above, but this bacterium was not isolated from the control plants. Phytosanitary measures must be taken to avoid spread of the pathogen to ornamentals in new landscape areas in Turkey. This report is important because infected Spirea spp. can be a potential inoculum source for other rosaceous ornamentals. To our knowledge, this is the first report of the occurrence of fire blight on meadowsweet in Turkey. References: (1) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992. (2) A. L. Jones and K. Geider. Laboratory Guide for Identification of Plant Pathogenic Bacteria, pp. 40-55. American Phytopathological Society, St. Paul, MN, 2001. (3) R. A. Lelliott and D. E. Stead. Methods for Diagnosis of Bacterial Diseases of Plants (Methods in Plant Pathology). Oxford, UK, 1987.

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High-efficient transgenic hairy roots induction in chicory: re-dawn of a traditional herb

Acta agriculturae Slovenica ◽

10.14720/aas.2016.107.2.06 ◽

2016 ◽

Vol 107 (2) ◽

pp. 321 ◽

Cited By ~ 2

Author(s):

Sara Kabirnataj ◽

Ghorbanali Nematzadeh ◽

Jafar Zolala ◽

Ahmad Farhad Talebi

Keyword(s):

Hairy Root ◽

Hairy Roots ◽

Biomass Accumulation ◽

Ms Medium ◽

Heterologous Proteins ◽

Bacterial Strains ◽

Root Cells ◽

Whole Process ◽

Cell Factories ◽

High Efficient

Plant roots can be manipulated by Agrobacterium rhizogenes to stimulate the production of heterologous proteins for pharmaceutical applications as green cell-factories. During the present study, four bacterial strains (A4, ATCC15834, ATCC11325 and A13) in combination with three co-cultivation media (MS, B5, LS) were examined to establish an efficient and reliable transformation system for chicory (Cichorium intybus L.) using A. rhizogenes. The maximum chicory hairy roots induction was achieved using A13 strain. The observation confirmed that MS medium was more effective on hairy root growth. Dried biomass accumulation of hairy roots infected by A13 strain was 1.10 g l-1 in MS medium which was significantly higher than those grown in LS and B5 medium (0.88 and 0.72 g l-1, respectively). Beta-glucuronidase (GUS) gene was introduced by A13 strain carrying the pCAMBIA1304 binary vector. The results showed that the highest frequency of transformation (63.15 %) was achieved using A13 strain and MS cultivation medium. Detection of GUS and hptII genes by PCR and GUS histochemical localization confirmed the integrative transformation in hairy roots. In conclusion, the whole process was successfully optimized as a pre-step to manipulate the chicory hairy root cells to improve the unique potential of secondary metabolite production.

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Production of Bacalin, Bacalein and Wogonin in Hairy Root Culture of American Skullcap (Scutellaria lateriflora)by Auxin Treatment

Biosciences Biotechnology Research Asia ◽

10.13005/bbra/2493 ◽

2017 ◽

Vol 14 (2) ◽

pp. 673-677 ◽

Cited By ~ 4

Author(s):

Do Yeon Kwon ◽

Haeng Hoon Kim ◽

Jong Seok Park ◽

Sang Un Park ◽

Nam Il Park

Keyword(s):

Hairy Root ◽

Hairy Roots ◽

Root Culture ◽

Hairy Root Culture ◽

Control Treatment ◽

Naphthaleneacetic Acid ◽

Hairy Root Cultures ◽

Root Cultures ◽

Alternative Approach ◽

Scutellaria Lateriflora

ABSTRACT: The hairy root culture of American Skullcap (Scutellaria lateriflora) was studied to investigate the biomass and flavonoids content (baicalin, baicalein and wogonin) in response of various auxin concentrations.The growth rates of the hairy roots varied significantly only at IBA 0.1 mg/L and for all other auxin treatments did not vary significantly. The biomass of hairy roots was 8% higher when treated with IBA 0.1 mg/L and biomass was almost similar and slightly lower levels when treated with various IAA concentration and NAA, respectively. However, the auxins treatments responsed positively to increase flavone production in American Skullcaphairy root culture. The auxin indole-3-butyric acid (IBA) at 1 mg/L performed the best for the accumulation of baicalin and wogonin. The auxin IBA at 1 mg/L accumulated 1.64 and 2.92 times higher baicalin and wogonin, respectively compared to control treatment. Meanwhile, the highest levels of baicalein were observed for hair root cultures in the presence of 1-naphthaleneacetic acid (NAA) at 0.1 mg/L achieving 2.38 times higher than that of accumulated in the control. These findings indicate that hairy root cultures of S. lateriflorausing liquid 1/2MS medium supplemented with auxin could be a valuable alternative approach for flavonoid production.

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First Report of Agrobacterium vitis as the Causal Agent of Grapevine Crown Gall in Serbia

Plant Disease ◽

10.1094/pdis-07-11-0600 ◽

2012 ◽

Vol 96 (2) ◽

pp. 286-286 ◽

Cited By ~ 2

Author(s):

N. Kuzmanović ◽

A. Ćalić ◽

M. Ivanović ◽

K. Gašić ◽

J. Pulawska ◽

...

Keyword(s):

Crown Gall ◽

Causal Agent ◽

Ammonium Citrate ◽

Ferric Ammonium Citrate ◽

23S Rrna ◽

Rrna Gene ◽

Agrobacterium Vitis ◽

First Report ◽

Crown Gall Disease ◽

Grapevine Crown Gall

In November 2010, a serious outbreak of crown gall disease was observed on 3-year-old grapevine (Vitis vinifera L.) cv. Cabernet Sauvignon grafted onto Kober 5BB rootstock in two commercial vineyards located in the South Banat District in Serbia. Large, aerial tumors were visible above the grafting point on grapevine trunks, and in most cases, the tumors completely girdled the trunk. From the gall tissues, white, circular, and glistening bacterial colonies were isolated on yeast mannitol agar medium. Eight, nonfluorescent, gram-negative, and oxidase-positive strains were isolated from seven tumor samples and selected for further identification. PCR assays with A/C′ (1) and VCF3/VCR3 (4) primers corresponding to the virD2 and virC genes yielded 224- and 414-bp fragments, respectively, confirming that the strains harbored the plasmid responsible for pathogenicity. The strains were differentiated to the species/biovar level with a multiplex PCR assay targeting 23S rRNA gene sequences (3) and were identified as Agrobacterium vitis. The 16S rDNA gene sequence from one isolate (GenBank Accession No. JN185718) showed 99% identity to the sequences of A. vitis previously deposited in NCBI GenBank database. The physiological and biochemical test results corresponded to the results of genetic analysis (2). The strains grew at 35°C and in nutrient broth supplemented with 2% NaCl. They were negative in 3-ketolactose, acid clearing on PDA supplemented with CaCO3, and ferric ammonium citrate tests; nonmotile at pH 7.0; pectolytic at pH 4.5; utilized citrate; produced acid from sucrose and alkali from tartarate. Pathogenicity was confirmed by inoculation of three plants per bacterial strain on grapevine cv. Cabernet Franc and on a local cultivar of tomato (Lycopersicon esculentum L.). The plants were inoculated on the stem by pricking one to three times through a drop of inoculum (108 CFU/ml) at three inoculation sites. Sterile distilled water was used as a negative control. Inoculated plants were maintained in a greenhouse at 24 ± 3°C. Typical tumors developed at the inoculation sites on tomatoes 3 weeks after inoculation and on grapevine 6 weeks after inoculation. No symptoms were observed on the control plants. Bacteria were reisolated from tumorigenic tissues and identified as pathogenic A. vitis by PCR. Crown gall disease was sporadically observed in vineyards in Serbia in previous years, but did not cause significant damage. Therefore, the causal agent was not studied in detail. To our knowledge, this is the first report of A. vitis determined as the causal agent of grapevine crown gall in Serbia. References: (1) J. H. Haas et al. Appl. Environ. Microbiol. 61:2879, 1995. (2) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) J. Pulawska et al. Syst. Appl. Microbiol. 29:470, 2006. (4) K. Suzaki et al. J. Gen. Plant Pathol. 70:342, 2004.

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Phases of crown-gall transformation susceptible to hydroxyurea

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1984.007 ◽

2014 ◽

Vol 53 (1) ◽

pp. 59-65 ◽

Cited By ~ 2

Author(s):

Aldona Rennert ◽

Józef Kowalczyk ◽

Halina Pajkowska

Keyword(s):

Agrobacterium Tumefaciens ◽

Crown Gall ◽

Pathogenic Bacteria ◽

Direct Action ◽

Plant Cells ◽

Induction Phase ◽

Bacterial Strains ◽

Tumour Formation ◽

Kalanchoë Daigremontiana ◽

Anti Tumour Effect

With the use of bacterial strains, both sensitive and resistant to hydroxyurea the action of this inhibitor on tumour formation on the leaves of Kalanchoe daigremontiana infected with Agrobacterium tumefaciens was tested for five days after inoculation. The results are in agreement with the opinion that the anti-tumour effect of hydroxyurea applied in the induction phase (between 18 and 60 h after inoculation) is the result of its direct action on plant cells, whereas inhibition of tumour formation by the inhibitor in the inoculation period depends on its action on the pathogenic bacteria.

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Identification of Agrobacterium vitis as a causal agent of grapevine crown gall in Serbia

Archives of Biological Sciences ◽

10.2298/abs1204487k ◽

2012 ◽

Vol 64 (4) ◽

pp. 1487-1494 ◽

Cited By ~ 2

Author(s):

N. Kuzmanovic ◽

Katarina Gasic ◽

M. Ivanovic ◽

Andjelka Prokic ◽

A. Obradovic

Keyword(s):

Crown Gall ◽

23S Rrna ◽

Vitis Vinifera L ◽

Rrna Gene ◽

Agrobacterium Vitis ◽

Biochemical Tests ◽

Crown Gall Disease ◽

Multiplex Pcr Assay ◽

Pcr Product ◽

23S Rrna Gene

In 2010, a serious outbreak of crown gall disease was observed on grapevines (Vitis vinifera L. cv. Cabernet Sauvignon) in several commercial vineyards located in the Vojvodina province, Serbia. Bacteria were isolated from the young tumor tissue on nonselective YMA medium and five representative strains were selected for further identification. Tumorigenic (Ti) plasmid was detected in all strains by PCR using primers designed to amplify the virC pathogenicity gene, producing a 414-bp PCR product. The strains were identified as Agrobacterium vitis using differential physiological and biochemical tests, and a multiplex PCR assay targeting 23S rRNA gene sequences. In the pathogenicity assay, all strains induced characteristic symptoms on inoculated tomato and grapevine plants. They were less virulent on tomato plants in comparison to the reference strains of A. tumefaciens and A. vitis.

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First Report of Pectobacterium carotovorum Causing Soft Rot of Opium Poppy in Spain

Plant Disease ◽

10.1094/pdis-92-2-0317a ◽

2008 ◽

Vol 92 (2) ◽

pp. 317-317 ◽

Cited By ~ 5

Author(s):

S. Aranda ◽

M. Montes-Borrego ◽

F. J. Muñoz-Ledesma ◽

R. M. Jiménez-Díaz ◽

B. B. Landa

Keyword(s):

Pathogenic Bacteria ◽

Soft Rot ◽

Opium Poppy ◽

Bacterial Suspension ◽

Fluorescent Light ◽

Pectobacterium Carotovorum ◽

Bacterial Strains ◽

First Report ◽

Biolog System ◽

Carotovorum Subsp

Opium poppy (Papaver somniferum L.) is an economically important pharmaceutical crop in Spain. Approximately 8,000 ha are cultivated annually in southern and central Spain. To improve yields, opium poppy cultivation is expanding to more humid or irrigated areas of Spain. In the springs of 2005 and 2007, we observed poppy plants with wilt and stem rot symptoms in irrigated, commercial opium poppy (cv. Nigrum) at Carmona and Écija, which are in Seville Province in southern Spain. Closer observations of affected plants revealed darkening and water soaking of the leaves and stem at the soil level, wilting of the lower leaves or the entire plant, and dark brown discoloration of stem vascular tissues and pith of the plant. Severely affected plants became completely rotten and collapsed. Isolations from symptomatic tissues on nutrient agar consistently yielded bacterial colonies. Pure cultures of four representative bacterial strains (two per each of affected field and year of isolation) were used in triplicate for a comparative analysis of biochemical and physiological traits in the ‘carotovora’ group of Erwinia (1) with known isolates of Pectobacterium carotovorum subsp. carotovorum, P. carotovorum subsp. atrosepticum, and Dickeya chrysanthemi. The isolates from opium poppy were gram negative, facultatively anaerobic, oxidase negative, catalase positive, grew at 37°C, and did not produce gas from D-glucose. Acid was produced from D(+)-arabinose, lactose, and D(+)-trehalose, but not from α-D-methylglucoside. In addition, the opium poppy bacterial isolates caused soft rot on potato slices within 24 h at 25°C and did not induce a hypersensitive reaction on tobacco leaves. Use of the Biolog GN microplates and the OmniLog ID 1.2 system identified the four poppy isolates as P. carotovorum (showing a 66.7% similarity with the subsp. carotovorum). Pathogenicity of poppy isolates was tested on three 6-week-old opium poppy plants (cv. Nigrum) by injecting 100 μl of a bacterial suspension containing 108 CFU/ml in the basal stem. Plants that served as controls were injected with sterile water. Plants were incubated in a growth chamber adjusted to 28°C, 90% relative humidity, and a 14-h photoperiod of fluorescent light of 360 μE·m-2·s-1. Severe symptoms of soft rot and darkening developed on stems of inoculated plants within 3 to 5 days after inoculation. No symptoms developed on control plants. Bacterial strains reisolated from inoculated plants were identified as P. carotovorum on the basis of the Biolog system, as well as biochemical and physiological characters. To our knowledge, this is the first report of P. carotovorum causing soft rot of commercial opium poppy crops in Spain and elsewhere. The presence of this bacterial pathogen to irrigated crops and humid areas may pose an important constraint on the yield of opium poppy crops in Spain. References: (1) R. S. Dickey and A. Kelman. Pages 44-59 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad, ed. The American Phytopathological Society, St. Paul, MN, 1988.

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First Report of Head Rot of Brassica oleracea convar. botrytis var. italica Caused by Pseudomonas fluorescens in Southern Italy

Plant Disease ◽

10.1094/pdis-91-5-0638a ◽

2007 ◽

Vol 91 (5) ◽

pp. 638-638 ◽

Cited By ~ 1

Author(s):

P. Lo Cantore ◽

N. S. Iacobellis

Keyword(s):

Pseudomonas Fluorescens ◽

Brassica Oleracea ◽

Pathogenic Bacteria ◽

Southern Italy ◽

Similarity Index ◽

Identification System ◽

Bacterial Strains ◽

First Report ◽

Pectolytic Activity ◽

Head Rot

During the winter of 2004, symptoms were observed in commercial cauliflower (Brassica oleracea L. convar. botrytis (L.) Alef. var. italica) fields of “romanesco” type (cv. Navona) in Apulia, southern Italy. These symptoms were noted on inflorescences that were almost ready for harvest, and a bacterial etiology was suspected. In particular, the corymbs showed water-soaked and brown discolored areas which then rotted. The above alterations involved the whole inflorescences, or in some cases, only a few florets. Longitudinal sections of the symptomatic inflorescences or the single floret showed brown discoloration and rotting of the internal tissues. The disease caused severe crop losses (approximately 100% either in the field or after harvest). Bacteria were isolated from water-soaked and soft-rotted cauliflower heads on King's medium B (KB). The strains were purified on nutrient agar and assayed for pathogenicity on subcorymbs from freshly harvested cauliflower heads. Bacterial suspensions containing approximately 108 CFU/ml were then sprayed on the surface of subcorymbs (3 subcorymbs per strain). Furthermore, in other pathogenicity assays, the florets were dipped in 108 CFU/ml bacterial suspensions or small aliquots of inoculum were injected into the peduncle of subcorymbs with a sterile syringe. Cauliflower heads treated with sterile distilled water were used as controls. After inoculation, the subcorymbs were maintained at 25°C and approximately 100% relative humidity for 48 h. All bacterial strains either applied to cauliflower subcorymbs by spray inoculation or dipping reproduced the disease symptoms. Intensity of symptoms varied with the inoculation method. Injection of bacteria caused water soaking and soft rot of cauliflower internal tissues. No symptoms were observed in negative control subcorymbs inoculated with sterile water. All bacterial strains were gram negative and fluorescent on KB. Isolates (17 of 18) showed the LOPAT characters of group Vb (++−+−) fluorescent pseudomonads, and only strain USB1237 showed characters of group IVb (−+++−) (3). The pectolytic activity of the latter strain was confirmed by the pectinase plate assay (4). The identity of representative strains was confirmed by the nutritional profile obtained with the Biolog Identification System (MicroLogTM System Release 4.2; Biolog, Inc., Hayward, CA). Strains USB1224, USB1226, USB1228, USB1231, USB1235, USB1236, USB1238, and USB1239 were identified as Pseudomonas fluorescens with similarity indices of 0.86, 0.52, 0.73, 0.81, 0.73, 0.74, 0.69, and 0.85, respectively. The pectolytic strain USB1237 was identified as a Pseudomonas spp. that is closely related to P. putida (similarity index = 0.45). In conclusion, the above results indicate that P. fluorescens is responsible for head rot of cauliflower. A similar disease has been previously reported on broccoli in different areas (1,2), but to our knowledge, this is the first report of head rot of cauliflower caused by P. fluorescens. References: (1) C. H. Canaday et al. Phytopathology 77:1712, 1987. (2) P. D. Hildebrand. Can. J. Plant Pathol. 8:350, 1986. (3) R. A. Lelliott and D. E. Stead. Methods for the diagnosis of bacterial diseases of plants. In: Methods in Plant Pathology. Vol. 2, T. F. Preece, ed. Blackwell Scientific Publications, Oxford, UK, 1987. (4) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, MN, 2001.

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