Pamela D. Roberts

Systematic analysis of xanthomonads (Xanthomonas spp.) associated with pepper and tomato lesions.

The taxonomy and evolutionary relationships among members of the genus Xanthomonas associated with tomato and pepper have been a matter of considerable controversy since their original description in 1921. These bacteria, which are a major affliction of tomato and pepper crops in warm and humid regions, were originally described as a single species, but subsequent research has shown the existence of at least two genetic groups differentiated by physiological, biochemical and pathological characteristics. This work synthesizes the findings from several approaches, including pathogenicity tests, enzymic activity, restriction fragment analysis of the entire genome, DNA-DNA hybridization and RNA sequence comparisons based on a 2097 base sequence comprising the 16S rRNA gene, the intergenic spacer located between the 16S and 23S rRNA genes and a small region of the 23S rRNA gene. Within the group of xanthomonads pathogenic on pepper and tomato four distinct phenotypic groups exist, of which three form distinct genomic species. These include Xanthomonas axonopodis pv. vesicatoria (A and C group), Xanthomonas vesicatoria (B group) and Xanthomonas gardneri (D group). On the basis of phenotypic and genotypic differences between A- and C-group strains, the C strains should be considered as a subspecies within Xanthomonas axonopodis pv. vesicatoria.

Recent genotypes of Phytophthora infestans in the eastern United States reveal clonal populations and reappearance of mefenoxam sensitivity.

Isolates of Phytophthora infestans (n = 178) were collected in 2002 to 2009 from the eastern United States, Midwestern United States, and eastern Canada. Multilocus genotypes were defined using allozyme genotyping, and DNA fingerprinting with the RG-57 probe. Several previously described and three new mulitilocus genotypes were detected. The US-8 genotype was found commonly on commercial potato crops but not on tomato. US-20 was found on tomato in North Carolina from 2002 through 2007 and in Florida in 2005. US-21 was found on tomato in North Carolina in 2005 and Florida in 2006 and 2007. US-22 was detected on tomato in 2007 in Tennessee and New York and became widespread in 2009. US-22 was found in 12 states on tomato and potato and was spread on tomato transplants. This genotype accounted for about 60% of all the isolates genotyped. The US-23 genotype was found in Maryland, Virginia, Pennsylvania, and Delaware on both tomato and potato in 2009. The US-24 genotype was found only in North Dakota in 2009. A1 and A2 mating types were found in close proximity on potato and tomato crops in Pennsylvania and Virginia; therefore, the possibility of sexual reproduction should be monitored. Whereas most individuals of US-8 and US-20 were resistant to mefenoxam, US-21 appeared to be intermediately sensitive, and isolates of US-22, US-23, and US-24 were largely sensitive to mefenoxam. On the basis of sequence analysis of the ras gene, these latter three genotypes appear to have been derived from a common ancestor. Further field and laboratory studies are underway using simple sequence repeat genotyping to monitor current changes in the population structure of P. infestans causing late blight in North America.

The 2009 late blight pandemic in the eastern United States - causes and results

The tomato late blight pandemic of 2009 made late blight into a household term in much of the eastern United States. Many home gardeners and many organic producers lost most if not all of their tomato crop, and their experiences were reported in the mainstream press. Some CSAs (Community Supported Agriculture) could not provide tomatoes to their members. In response, many questions emerged: How did it happen? What was unusual about this event compared to previous late blight epidemics? What is the current situation in 2012 and what can be done? Its easiest to answer these questions, and to understand the recent epidemics of late blight, if one knows a bit of the history of the disease and the biology of the causal agent, Phytophthora infestans.

Characterization of Phytophthora capsici Associated with Roots of Weeds on Florida Vegetable Farms

Weeds were sampled in commercial vegetable fields in Palm Beach County, FL in August 2001, December 2001, and March 2002 for the presence of Phytophthora capsici. Fields sampled had a recent history of this plant pathogen. P. capsici was successfully isolated from the roots of six of 42 Carolina geranium (Geranium carolinianum) plants, four of 28 American black nightshade (Solanum americanum) plants, and two of 130 common purslane (Portulaca oleracea) plants. All but one of the 12 isolates were of the A1 mating type. All 12 isolates were resistant to mefenoxam, although at different levels. All but one isolate were strongly pathogenic on pepper seedlings. When two or three isolates recovered from each weed were inoculated onto the roots of their weed host of origin, P. capsici was recovered from the roots. Isolates of P. capsici were tested on four other solanaceous weeds of importance or potential importance to agricultural fields in Florida: Solanum nigrum, S. ptycanthum, S. carolinense, and S. capsicoides. Recovery of P. capsici from roots varied with weed species and isolate tested. P. capsici caused disease mortality on S. nigrum, and no reisolation of P. capsici was possible with S. capsicoides. This is the first report of S. americanum and G. carolinianum being alternative hosts for P. capsici under field conditions. This study also validated P. oleracea as an alternative host. In Florida, and perhaps elsewhere, weeds may contribute to pathogen survival in the absence of a host crop or when propagules may not readily survive in soil or plant debris.

Survival of Xanthomonas fragariae on strawberry in summer nurseries in Florida detected by specific primers and nested polymerase chain reaction.

Genomic DNA from strain XF1425 of Xanthomonas fragariae was amplified with primers RST2 and RST3 from the hrp-gene region of Xanthomonas campestris pv. vesicatoria. The polymerase chain reaction (PCR) product was sequenced. Four primers were selected at sites unique to X. fragariae, which were identified by comparison with the sequences of PCR products amplified by the same primers from DNA of three strains of X. campestris pv. vesicatoria. Three primers were specific for amplification of DNA from X. fragariae but not from strains of 16 pathovars of X. campestris or nonpathogenic xanthomonads from strawberry. Bacteria were detected at approximately 10 4 to 10 5 CFU/ml by a single round of PCR. A nested PCR technique enabled detection to approximately 18 cells. Restriction endonuclease digestion patterns of the PCR product were unique to X. fragariae and confirmed amplification of DNA from the target organism. Bacteria were detected from symptomatic and asymptomatic plant tissue by the nested technique. From strawberry plants inoculated with a rifampicin-resistant strain of X. fragariae and planted in the field in Florida, bacteria were detected by nested PCR and by recovery onto media at 2-week intervals for 92 days after planting. Daughter plants of the inoculated plants were positive for X. fragariae by nested PCR amplification, indicating that X. fragariae survived on plants in summer nurseries in Florida and was disseminated to daughter plants.

Identification and Characterization of a Novel Whitefly-Transmitted Member of the Family Potyviridae Isolated from Cucurbits in Florida

ABSTRACT A novel whitefly-transmitted member of the family Potyviridae was isolated from a squash plant (Cucurbita pepo) with vein yellowing symptoms in Florida. The virus, for which the name Squash vein yellowing virus (SqVYV) is proposed, has flexuous rod-shaped particles of approximately 840 nm in length. The experimental host range was limited to species in the family Cucurbitaceae, with the most dramatic symptoms observed in squash and watermelon, but excluded all tested species in the families Amaranthaceae, Apocynaceae, Asteraceae, Chenopodiaceae, Fabaceae, Malvaceae, and Solanaceae. The virus was transmitted by whiteflies (Bemisia tabaci) but was not transmitted by aphids (Myzus persicae). Infection by SqVYV induced inclusion bodies visible by electron and light microscopy that were characteristic of members of the family Potyviridae. Comparison of the SqVYV coat protein gene and protein sequences with those of recognized members of the family Potyviridae indicate that it is a novel member of the genus Ipomovirus. A limited survey revealed that SqVYV also was present in watermelon plants suffering from a vine decline and fruit rot recently observed in Florida and was sufficient to induce these symptoms in greenhouse-grown watermelon, suggesting that SqVYV is the likely cause of this disease.

A Non-Hypersensitive Resistance in Pepper to the Bacterial Spot Pathogen Is Associated with Two Recessive Genes

ABSTRACT The pepper genotype, ECW-12346, was developed with bacterial spot resistance derived from Pep13, PI 271322, and ECW123 (Early Calwonder containing Bs1, Bs2, and Bs3 genes). For genetic analysis of this resistance, ECW12346, ECW123, F(1), F(2), and backcrosses were inoculated with a pepper race 6 (P6) strain. Two recessive genes were identified that determined resistance. The genes are designated bs5 and bs6 for the resistance derived from PI 271322 and Pep13, respectively. In greenhouse and field studies, ECW12346 was highly resistant, whereas ECW123 had significant defoliation. In growth-room studies, electrolyte leakage and population dynamics were determined. Following infiltration of both genotypes with 10(8) CFU/ml of a P6 strain, there was no rapid increase in electrolyte leakage within 72 h, whereas a rapid increase in electrolyte leakage occurred within 24 h when a similar concentration of a P3 strain (containing the avrBs2 gene) was infiltrated into the intercellular spaces of the leaf. When 10(5) CFU/ml of a P6 strain was infiltrated into leaves, complete tissue collapse was evident in ECW123 10 days later as determined by visual assessment and electrolyte leakage data, but no confluent necrosis was detected in ECW12346. Internal populations were at least two logarithmic units higher in ECW123 than in ECW12346. Therefore, ECW12346 inhibits population build-up without inducing the typical hypersensitive reaction characterized by an increase in electrolyte leakage.

Relative Importance of Bacteriocin-Like Genes in Antagonism of Xanthomonas perforans Tomato Race 3 to Xanthomonas euvesicatoria Tomato Race 1 Strains

ABSTRACT In a previous study, tomato race 3 (T3) strains of Xanthomonas perforans became predominant in fields containing both X. euvesicatoria and X. perforans races T1 and T3, respectively. This apparent ability to take over fields led to the discovery that there are three bacteriocin-like compounds associated with T3 strains. T3 strain 91-118 produces at least three different bacteriocin-like compounds (BCN-A, BCN-B, and BCN-C) antagonistic toward T1 strains. We determined the relative importance of the bacteriocin-like compounds by constructing the following mutant forms of a wild-type (WT) T3 strain to evaluate the antagonism to WT T1 strains: Mut-A (BCN-A−), Mut-B (BCN-B−), Mut-C (BCN-C−), Mut-AB, Mut-BC, and Mut-ABC. Although all mutant and WT T3 strains reduced the T1 populations in in planta growth room experiments, Mut-B and WT T3 were significantly more effective. Mutants expressing BCN-B and either BCN-A or BCN-C reduced T1 populations less than mutants expressing only BCN-A or BCN-C. The triple-knockout mutant Mut-ABC also had a significant competitive advantage over the T1 strain. In pairwise-inoculation field experiments where plants were coinoculated with an individual mutant or WT T3 strain and the T1 strain, the mutant strains and the WT T3 strain were reisolated from more than 70% of the lesions. WT T3 and Mut-B were the most frequently reisolated strains. In field experiments where plants were group inoculated with Mut-A, Mut-B, Mut-C, Mut-ABC, and WT T1 and T3 strains, Mut-B populations dominated all three seasons. In greenhouse and field experiments, the WT and mutant T3 strains had a selective advantage over T1 strains. Bacterial strains expressing both BCN-A and BCN-C appeared to have a competitive advantage over all other mutant and WT strains. Furthermore, BCN-B appeared to be a negative factor, with mutant T3 strains lacking BCN-B having a selective advantage in the field.

Disease Progress, Yield Loss, and Control of Xanthomonas fragariae on Strawberry Plants

The progress of angular leaf spot, caused by the bacterium Xanthomonas fragariae, was examined in field plots of strawberry in the 1994 and 1995 seasons. Disease severity increased intermittently to maxima of circa 25% in 1994 and 15% in 1995. Angular leaf spot reduced marketable yield 8.6% in 1994 and 7.7% in 1995, despite differences in disease severity and base marketable yields for the two seasons. Minimal spread of the pathogen occurred from field plots with inoculated plants to plots with non-inoculated plants. A mixture of cupric hydroxide plus mancozeb was applied at the label rate (1×) at 7- to 14-day intervals and at a reduced rate (0.1×) at 2- to 4-day intervals. The bactericidal mixture at the 1× rate significantly decreased disease, but this mixture was phytotoxic; both plant size and yield were reduced. The 0.1× rate was nonphytotoxic and it reduced disease severity in both years and increased yield in 1994. Lesions of angular leaf spot were detected on strawberry transplants imported from nurseries in Canada and northern United States in both 1993 and 1994. X. fragariae was isolated from those lesions.

First Report of Downy Mildew Caused by a Peronospora sp. on Basil in Florida and the United States

Basil is grown as a specialty crop in greenhouse and field production in Florida and other regions of the United States. Downy mildew on basil (Ocimum basilicum) was detected from four production sites (Collier, Hendry, Miami-Dade, and Palm Beach counties) in south Florida in the fall of 2007, and within months, it was also found in west-central north Florida (Hillsborough County). Incidence reached nearly 100% on some of the affected crops and caused complete yield losses on basil grown both in the field for fresh market and potted herbs market. Symptoms developed during transit on basil that appeared symptomless at harvest. Symptoms initially appeared as yellowing on the lower leaves that was typically delineated by the veins, although in some cases the entire leaf area of the leaf surface was affected. A gray, fuzzy growth was apparent on the abaxial leaf surface. Microscopic observation detected dichotomous branching, hyaline sporangiophores (220 to 750 × 4 to 9 μm) bearing single sporangia. Sporangia were light brown, ovoid to slightly ellipsoid, and measured 14 to 15 × 15 to 18 μm. Oospores were not observed. Leaves of potted basil plants and coleus (Solenostemon scutellarioides) were inoculated with a suspension containing 1 × 105 sporangia/ml and sprayed till runoff (approximately 15 ml per plant) with a hand-held pressurized aerosol canister. Plants were covered with a plastic bag for 24 h and maintained in the greenhouse under ambient conditions. Noninoculated plants served as controls. After 7 days, symptoms typical of downy mildew occurred only on the inoculated basil plants and sporulation was confirmed microscopically. The internal transcribed spacer regions of an isolate collected in Hendry County were sequenced bidirectionally. The consensus sequence was deposited into GenBank (Accession No. FJ346561). Sequence data matched (100% homology) with a Peronospora sp. reported on sweet basil in Switzerland (GenBank Accession No. AY884605) and was similar (99% homology) to an isolate (GenBank Accession No. DQ523586) reported on coleus, although inoculation to coleus failed to confirm pathogenicity on this host. The sequence data also distinguished the isolate from P. lamii (87% homology) previously reported to occur on basil. The pathogen was identified as a Peronospora sp. based on morphological characteristics and sequencing homology (1-3). References: (1) L. Belbahri et al. Mycol. Res. 109:1276, 2005. (2) S. Francis. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 688. CMI, Kew, England, 1981. (3) A. McLeod et al. Plant Dis. 90:1115, 2006.