First Report of Cylindrocarpon sp. Associated with Root Rot Disease of Strawberry in North Carolina.

Strawberry (Fragaria × ananassa Duchesne) is an economically important fruit crop in North Carolina for domestic consumption and export. In April 2012, outbreaks of a destructive root disease were observed in strawberry cv. Chandler in Buncombe, New Hanover, and Roman counties, North Carolina. Samples from Rowan (ID 13175) and Buncombe (ID 13193) counties submitted to the Plant Disease and Insect Clinic of the Department of Plant Pathology, North Carolina State University, exhibited yellowing and wilting of leaves and extensive root necrosis, and disease severity based on field symptoms ranged from 20 to 30%. To identify the pathogen, five small pieces of necrotic crown and root tissues were taken from each sample, surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, and plated onto potato dextrose agar (PDA) with 0.5 g liter-1 of streptomycin sulfate. Colonies developing from the tissue samples were transferred to PDA. Colonies from both samples were identical, grew relatively slowly, and gradually turned yellowish to partially brownish. After about 7 days, abundant conidia were formed. These were hyaline, mostly straight with both ends rounded, predominantly three septate, and 40 to 50 × 5 to 10 µm. Based on morphological characteristics, these isolates were identified as a species of Cylindrocarpon (1) To confirm the original identification of the fungus as a species of Cylindrocarpon, genomic DNA of both isolates was extracted from mycelia using DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA) and analyzed using PCR (2). The internal transcribed spacers (ITS)1 and (ITS)2 flanking the 5.8S rRNA regions were amplified and sequenced using universal primers ITS1 (forward) and ITS4 (reverse). The sequences of the 421 bp (GenBank KC847090 and KC847091) of both isolates were identical. Furthermore, a BLASTn search of these sequences showed homology of 99% with the sequences of Cylidrocarpon species (AB369421.1, AM419069.1, AM419074.1, AY295332.1, JN031017.1, JN253505.1, and JQ886422.1), To fulfill Koch's postulates, inoculum of each isolate was prepared and adjusted to 1.5 × 107 conidia/ml using a hemacytometer. 'Chandler' strawberry plants were grown in 25-cm diameter plastic pots (one seedling per pot) in the greenhouse and five 6-week-old plants were injected with conidia of each isolate into the base of crown using a 5-ml syringe. The plants were covered with clear plastic for 24 h and left on the greenhouse bench with a 16-h photoperiod and 25/20°C day/night temperatures and assessed for disease development 14 days after inoculation. The inoculated plants exhibited wilting and root necrosis, consistent with the symptoms observed on strawberry plants in the field. Control plants treated with distilled water remained healthy. Isolations were made from the inoculated plants and the fungus used for inoculation was recovered from all plants. The morphology of these isolates was in agreement with published descriptions of Cylindrocarpon (1). To our knowledge, this is the first report of a Cylindrocarpon sp. causing crown and root rot on strawberry in North Carolina and effective disease management strategies need to be explored. References: (1) C. D. Booth. Mycol. Pap. (CMI) 104:1, 1996. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Grafting Tomato to Manage Bacterial Wilt Caused by Ralstonia solanacearum in the Southeastern United States.

Bacterial wilt, caused by Ralstonia solanacearum, can result in severe losses to tomato (Solanum lycopersicum) growers in the southeastern United States, and grafting with resistant rootstocks may be an effective strategy for managing this disease. However, R. solanacearum populations maintain considerable diversity, and little information is known regarding the efficacy of commercially available rootstocks to reduce bacterial wilt incidence and subsequent crop loss in the United States. In this study, tomato plants grafted onto 'Dai Honmei' and 'RST-04-105-T' rootstocks had significantly lower area under the disease progress curve (AUDPC) values compared with nongrafted plants (P < 0.05). Across three locations in North Carolina, final bacterial wilt incidence for non- and self-grafted plants was 82 ± 14 to 100%. In contrast, bacterial wilt incidence for the grafted plants was 0 to 65 ± 21%. Final bacterial wilt incidence of plants grafted with Dai Honmei rootstock was 0 and 13 ± 3% at two locations in western North Carolina but 50 ± 3% at a third site in eastern North Carolina. Similarly, grafting onto RST-04-105-T rootstock significantly reduced AUDPC values at two of the three locations (P < 0.05) compared with that of the nongrafted plants, but performed poorly at the third site. Total fruit yields were significantly increased by grafting onto resistant rootstocks at all three sites (P < 0.05). Regression analyses indicated that yield was significantly negatively correlated with bacterial wilt AUDPC values (R2 was 0.4048 to 0.8034), and the use of resistant rootstocks enabled economically viable tomato production in soils naturally infested with R. solanacearum.

First Report of Anthracnose Caused by Colletotrichum fragariae on Cyclamen in North Carolina.

In November 2009, cyclamen (Cyclamen persicum) plants with disease symptoms from a commercial greenhouse operation in the western part of North Carolina were sent to the Plant Diseases and Insect Clinic at North Carolina State University. Symptoms consisted of coalescing reddish and tan necrotic leaf spots with concentric circles. Other symptoms included darkened vascular tissue and decay of the corm, large roots, and petioles. Diseased leaves and stems were surface sterilized in 0.5% sodium hypochlorite for 3 min, air dried, and placed in petri dishes containing alkaline water agar. After 3 days of incubation at room temperature, fungal colonies were transferred to acidified potato dextrose agar. Isolation frequency after 5 days was 33% (three of nine pieces) and 16% (one of six pieces) from small leaf spots and petioles, respectively. Pure cultures of isolates were gray and black with abundant, aerial, gray whitish mycelia. Diseased plants were also incubated in a moist chamber at room temperature and sporulation was observed within 7 days. Conidia were tapered with rounded ends and produced in the acervulus and on the tips of setae, which is consistent with the morphology of described isolates of Colletotrichum fragariae. Similar setae were also observed directly on the fine roots of the original sample. The pathogenicity of single-spore cultures was tested by spraying four 2-month-old cyclamen plants with a conidial suspension (106 conidia/ml) and the plants were kept in a humid chamber for 24 h. Noninoculated controls (four plants) were sprayed with distilled water and subjected to the same conditions. The pathogenicity test was also repeated. Inoculated plants and controls were placed in a greenhouse with a temperature range from 22 to 25°C. After 7 to 10 days, symptomatic leaves and stems were observed on all the inoculated plants but not on the control plants. Fungi reisolated from 10 symptomatic leaf tissues had identical morphological features as the original isolates. Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits following the manufacturer's protocol (Qiagen Inc., Valencia, CA). Sequence analysis of the rRNA internal transcribed spacer (ITS) region of the cyclamen isolate (GenBank Accession No. HQ188923), based on the fragment amplified with ITS1 and ITS4 primers, showed 100% similarity to isolates of C. fragariae deposited in GenBank (Accession Nos. FJ172290 [ATCC MYA-4443 from cyclamen] and FJ810510 [ATCC MYA-4442 from silver date palm]) and Florida isolate C16 isolated from strawberry (1). In addition, the morphology and ITS sequences of the cyclamen isolate were identical to those of the C. fragariae voucher isolate from strawberry (GU174546). Results from disease symptoms, colony and spore morphology, pathogenicity tests, and ITS sequence analysis suggest that C. fragariae was the pathogen responsible for the disease symptoms on cyclamens. To our knowledge, this is the first report of a disease caused by C. fragariae on cyclamen in North Carolina and complements an earlier report from Florida (1). Reference: (1) S. J. MacKenzie et al. Plant Dis. 92:1432, 2008.

Grafting Tomato with Interspecific Rootstock to Manage Diseases Caused by Sclerotium rolfsii and Southern Root-Knot Nematode.

Southern blight (Sclerotium rolfsii) and root-knot nematodes (Meloidogyne spp.) cause severe damage to fresh-market tomato (Solanum lycopersicum) throughout the southeastern United States. Grafting is an emerging technology in U.S. tomato production, and growers require information regarding the resistance characteristics conferred by rootstocks. In this study, southern blight (SB) and root-knot nematodes (RKN) were effectively managed using interspecific hybrid rootstocks. During 2007 and 2008, field trials were carried out at two locations that had soils naturally infested with S. rolfsii. At the end of the growing seasons, the mean SB incidence of nongrafted plants was 27 and 79% at the two sites. SB incidence among plants grafted onto rootstock cultivars Big Power (one location only), Beaufort, and Maxifort ranged from 0 to 5%, and area under the disease progress curve (AUDPC) values were lower than for nongrafted and self-grafted controls (P < 0.01). At one location, soils were naturally infested with RKN, and all three rootstocks reduced RKN AUDPC and RKN soil populations at first harvest (P < 0.01). Big Power was particularly effective at reducing RKN galling and RKN soil populations at final fruit harvest (P < 0.01). Fruit yield was higher when resistant rootstocks were utilized (P < 0.05), and in our study grafting was effective at maintaining crop productivity in soils infested with S. rolfsii and M. incognita.

Classification and Identification of Xanthomonas translucens Isolates, Including Those Pathogenic to Ornamental Asparagus.

ABSTRACT In order to confirm and refine the current classification scheme of Xanthomonas translucens and to identify novel strains from ornamental asparagus, a collection of field and reference strains was analyzed. Rep-polymerase chain reaction (PCR) genomic fingerprint profiles were generated from 33 isolates pathogenic to asparagus as well as 61 X. trans-lucens reference strains pathogenic to cereals and grasses. Amplified ribo-somal gene restriction analysis profiles were obtained from most of these and 29 additional Xanthomonas reference strains. Rep-PCR genomic fingerprint profiles of all strains were compared with those in a large Xanthomonas database using computer-assisted analysis. Rep-PCR ge-nomic fingerprinting facilitated the characterization and discrimination of X. translucens, including the pathovars arrhenatheri, graminis, phlei, phleipratensis, and poae, as well as a number of strains received as X. translucens pv. cerealis. Strains received as pathovars hordei, secalis, translucens, undulosa, and other cerealis strains were grouped in two subclusters that correspond to the recently redefined pathovars X. trans-lucens pvs. undulosa and translucens. All 33 novel isolates from ornamental asparagus (tree fern; Asparagus virgatus) were identified as X. translucens pv. undulosa. Moreover, a unique amplified small subunit ribosomal gene MspI/AluI restriction profile specific for all X. translucens strains tested, including those pathogenic to asparagus, allowed discrimination from all other Xanthomonas spp. Although phage tests were inconclusive, the classification of the asparagus strains within the X. translucens complex was supported by pathogenicity assays in which all the isolates from ornamental asparagus induced watersoaking on wheat. Surprisingly, several X. translucens reference strains affected asparagus tree fern as well. That the novel asparagus isolates belong to X. translucens pv. undulosa is extraordinary because all hosts of X. translucens pathovars described to date belong only to the families Gramineae and Poaceae, whereas asparagus belongs to the phylogenetically distant family Liliaceae.

A comprehensive species to strain taxonomic framework for xanthomonas.

ABSTRACT A comprehensive classification framework was developed that refines the current Xanthomonas classification scheme and provides a detailed assessment of Xanthomonas diversity at the species, subspecies, pathovar, and subpathovar levels. Polymerase chain reaction (PCR) using primers targeting the conserved repetitive sequences BOX, enterobacterial repetitive intergenic consensus (ERIC), and repetitive extragenic palindromic (REP) (rep-PCR) was used to generate genomic fingerprints of 339 Xanthomonas strains comprising 80 pathovars, 20 DNA homology groups, and a Stenotrophomonas maltophilia reference strain. Computer-assisted pattern analysis of the rep-PCR profiles permitted the clustering of strains into distinct groups, which correspond directly to the 20 DNA-DNA homology groups(genospecies) previously identified. Group 9 strains (X. axonopodis) were an exception and did not cluster together into a coherent group but comprised six subgroups. Over 160 strains not previously characterized by DNA-DNA hybridization analysis, or not previously classified, were assigned to specific genospecies based on the classification framework developed. The rep-PCR delineated subspecific groups within X. hortorum, X. arboricola, X. axonopodis, X. oryzae, X. campestris, and X. translucens. Numerous taxonomic issues with regard to the diversity, similarity, redundancy, or misnaming were resolved. This classification framework will enable the rapid identification and classification of new, novel, or unknown Xanthomonas strains that are pathogenic or are otherwise associated with plants.

Field Control of Bacterial Spot and Bacterial Speck of Tomato Using a Plant Activator.

Acibenzolar-S-methyl (CGA 245704 or Actigard 50WG) is a plant activator that induces systemic acquired resistance (SAR) in many different crops to a number of pathogens. Acibenzolar-S-methyl was evaluated for management of bacterial spot (Xanthomonas axonopodis pv. vesicatoria) and bacterial speck (Pseudomonas syringae pv. tomato) of tomato in 15 and 7 field experiments, respectively. Experiments were conducted over a 4-year period in Florida, Alabama, North Carolina, Ohio, and Ontario using local production systems. Applied at 35 g a.i. ha-1, acibenzolar-S-methyl reduced foliar disease severity in 14 of the 15 bacterial spot and all 7 bacterial speck experiments. Disease control was similar or superior to that obtained using a standard copper bactericide program. Acibenzolar-S-methyl also reduced bacterial fruit spot and speck incidence. Tomato yield was not affected by using the plant activator in the field when complemented with fungicides to manage foliar fungal diseases, but tomato transplant dry weight was negatively impacted. X. axonopodis pv. vesicatoria population densities on greenhouse-grown tomato transplants were reduced by acibenzolar-S-methyl treatment. Bacterial speck and spot population densities on leaves of field-grown plants were not dramatically affected. Acibenzolar-S-methyl can be integrated as a viable alternative to copper-based bactericides for field management of bacterial spot and speck, particularly where copper-resistant populations predominate.

Comparison of AFLP and rep-PCR genomic fingerprinting with DNA-DNA homology studies: Xanthomonas as a model system.

The genus Xanthomonas contains a large number of strains, which have been characterized by a variety of phenotypic and genotypic classification methods. The Xanthomonas collection constitutes one of the largest groups of bacteria that have been characterized phylogenetically by DNA-DNA homology studies and genomic fingerprinting. Presently, a total genomic DNA-DNA homology value of 70% represents an internationally accepted criterion to define bacterial species levels. However, the complexity of DNA-DNA reassociation kinetics methods precludes the rapid analysis of large numbers of bacterial isolates, which is imperative for molecular microbial diversity studies. Therefore, the aim of this study was to compare more facile PCR-based genomic fingerprinting techniques, such as repetitive-sequence-based (rep)-PCR and AFLP genomic fingerprinting, to DNA-DNA hybridization studies. Using three different primer sets, rep-PCR genomic fingerprint patterns were generated for 178 Xanthomonas strains, belonging to all 20 previously defined DNA-DNA homology groups, and one Stenotrophomonas maltophilia strain. In addition, AFLP genomic fingerprints were produced for a subset of 80 Xanthomonas strains belonging to the 20 DNA-DNA homology groups and for the S. maltophilia strain. Similarity values derived from rep-PCR- and AFLP-generated fingerprinting analyses were calculated and used to determine the correlation between rep-PCR- or AFLP-derived relationships and DNA-DNA homology values. A high correlation was observed, suggesting that genomic fingerprinting techniques truly reveal genotypic and phylogenetic relationships of organisms. On the basis of these studies, we propose that genomic fingerprinting techniques such as rep-PCR and AFLP can be used as rapid, highly discriminatory screening techniques to determine the taxonomic diversity and phylogenetic structure of bacterial populations.

Multiphasic analysis of xanthomonads causing bacterial spot disease on tomato and pepper in the Caribbean and central america: evidence for common lineages within and between countries.

ABSTRACT Four hundred thirty-three xanthomonad strains isolated from tomato or pepper plants from 32 different fields in four Caribbean and Central American countries were screened for the ability to hydrolyze starch and sodium polypectate and for resistance to copper and streptomycin. Of these, 95 representative strains were further characterized by various phnetic tests, and 63 of these strains were then analyzed by genomic fingerprinting. Most of the strains (>90%) were tolerant to copper. However, there was much more variability in sensitivity to streptomycin. All strains in Guadeloupe and 93% of the strains in Barbados were sensitive to streptomycin. The majority of strains were typical Xanthomonas campestris pv. vesicatoria group A strains. In Barbados, however, a unique group of strains was identified that was serologically similar to group A strains but was amylolytic. These strains were designated A1. The occurrence of X. campestris pv. vesicatoria group B strains in Central America was found to be limited to two fields in Costa Rica and one in Guatemala. No group B strains were identified in the Caribbean, in contrast to common occurrence in the central United States and in South America. T3 strains were not found in this study, despite the recent increase of such strains in Florida and Mexico. Unique strains from Costa Rica belonging to the X. gardneri group were identified. Little linkage was found among phenotypic and rep-polymerase chain reaction (rep-PCR) genomic fingerprinting profiles of the pathogens except at the species/pathovar level; strains displaying virtually identical fingerprint profiles were found to correspond to distinct races and vice versa. The rep-PCR genomic fingerprinting analyses suggest that certain lineages may have evolved or predominated in specific regions or specific countries.

rep-PCR-Mediated Genomic Fingerprinting: A Rapid and Effective Method to Identify Clavibacter michiganensis.

ABSTRACT The genomic DNA fingerprinting technique known as repetitive-sequence-based polymerase chain reaction (rep-PCR) was evaluated as a tool to differentiate subspecies of Clavibacter michiganensis, with special emphasis on C. michiganensis subsp. michiganensis, the pathogen responsible for bacterial canker of tomato. DNA primers (REP, ERIC, and BOX), corresponding to conserved repetitive element motifs in the genomes of diverse bacterial species, were used to generate genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. sepedonicus, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, and C. michiganensis subsp. insidiosum. The rep-PCR-generated patterns of DNA fragments observed after agarose gel electrophoresis support the current division of C. michiganensis into five subspecies. In addition, the rep-PCR fingerprints identified at least four types (A, B, C, and D) within C. michiganensis subsp. michiganensis based on limited DNA polymorphisms; the ability to differentiate individual strains may be of potential use in studies on the epidemiology and host-pathogen interactions of this organism. In addition, we have recovered from diseased tomato plants a relatively large number of naturally occurring avirulent C. michiganensis subsp. michiganensis strains with rep-PCR fingerprints identical to those of virulent C. michiganensis subsp. michiganensis strains.

Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR.

DNA primers corresponding to conserved motifs in bacterial repetitive (REP, ERIC, and BOX) elements and PCR were used to show that REP-, ERIC-, and BOX-like DNA sequences are widely distributed in phytopathogenic Xanthomonas and Pseudomonas strains. REP-, ERIC, and BOX-PCR (collectively known as rep-PCR) were used to generate genomic fingerprints of a variety of Xanthomonas and Pseudomonas isolates and to identify pathovars and strains that were previously not distinguishable by other classification methods. Analogous rep-PCR-derived genomic fingerprints were generated from purified genomic DNA, colonies on agar plates, liquid cultures, and directly from lesions on infected plants. REP, ERIC, and BOX-PCR-generated fingerprints of specific Xanthomonas and Pseudomonas strains were found to yield similar conclusions wtih regard to the identity of and relationship between these strains. This suggests that the distribution of REP-, ERIC, and BOX-like sequences in these strains is a reflection of their genomic structure. Thus, the rep-PCR technique appears to be a rapid, simple, and reproducible method to identify and classify Xanthomonas and Pseudomonas strains, and it may be a useful diagnostic tool for these important plant pathogens.