Rapid Detection of the Fusarium oxysporum Lineage Containing the Canary Island Date Palm Wilt Pathogen.

ABSTRACT Fusarium oxysporum f. sp. canariensis causes Fusarium wilt disease on the Canary Island date palm (Phoenix canariensis). To facilitate disease management, a polymerase chain reaction diagnostic method has been developed to rapidly detect the pathogen. A partial genomic library of F. oxysporum f. sp. canariensis isolate 95-913 was used to identify a DNA sequence diagnostic for a lineage containing all tested isolates of F. oxysporum f. sp. canariensis. Two oligonucleotide primers were designed and used to amplify a 567-bp fragment with F. oxysporum f. sp. canariensis DNAs. DNA from 61 outgroup isolates did not amplify using these primers. Once the primers were shown to amplify a 0.567-kb fragment from DNA of all the F. oxysporum f. sp. canariensis isolates tested, a rapid DNA extraction procedure was developed that led to the correct identification of 98% of the tested F. oxysporum f. sp. canariensis isolates.

First Report of Tomato Yellow Leaf Curl Virus in Tomato in South Georgia.

In October 1998, symptoms characteristic of tomato yellow leaf curl virus (TYLCV) were observed on fresh market tomato (Lycopersicon esculentum Mill.) in four production fields, two in Decatur County, Georgia, and two in Gadsden County, Florida. Symptoms observed were plant stunting, reduced leaf size, yellow leaf margins, and mottling. The incidence of symptomatic plants was less than 1% in all fields examined. In most cases, symptoms were observed only on the upper portion of plants, suggesting these plants had been infected by secondary spread from an unknown source. Nuclear inclusions characteristic of geminiviruses were observed by light microscopy in leaf tissue from symptomatic plants (1). To identify the geminivirus, total DNA from infected plants was extracted from six symptomatic tomato plants (two from Georgia and four from Florida) for polymerase chain reaction (PCR; J. E. Polston, personal communication). DNA was amplified with geminivirus DNA A degenerate primer set PAL1v1978 and PAR1c496 (2) from these extracts in addition to extracts from a known TYLCV-infected, a tomato mottle virus (ToMoV)-infected, and a healthy tomato plant. A PCR product of 1.4 kb was obtained from plants with TYLCV-like symptoms, while a 1.4-kb product and a 1.1-kb product were obtained from extracts of the known TYLCV-infected and ToMoV-infected tomato plants, respectively. No PCR product was obtained from extracts of healthy tomato plants. The 1.4-kb PCR products from one Georgia sample and one Florida sample were compared with those of TYLCV and ToMoV by restriction enzyme (RE) digestion with EcoRI and ClaI. The RE pattern of the 1.4-kb fragment from both samples was identical to the RE pattern of TYLCV and different from that of ToMoV. Adult and immature whiteflies collected from the fields where TYLCV was found were identified as Bemisia tabaci, the vector of TYLCV, but the biotype was not established. This report of TYLCV in south Georgia and north Florida extends the geographic range of TYLCV in the U.S. northward approximately 100 km. Georgia is the second state in which TYLCV was found since its initial detection in south Florida in July 1997 (J. E. Polston, personal communication). Monitoring of silverleaf whitefly populations and detection of TYLCV on alternate hosts will continue in order to estimate the potential impact of this virus on south Georgia and north Florida agriculture. References: (1) R. G. Christie and J. R. Edwardson. Plant Dis. 70:273, 1986, (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Partial Characterization of a Distinct Potyvirus Isolated from Watermelon in Florida.

Conspicuous, unusual nuclear inclusions in stained epidermal strips of leaves implicated a virus (designated isolate 2932) as the cause of foliar mosaic in a watermelon plant (Citrullus lanatus) received for analysis from South Florida in 1990. In greenhouse tests, mechanically inoculated plants of Cucurbita pepo (Small Sugar pumpkin and Early Prolific Straightneck squash) and watermelon (Crimson Sweet) developed mosaic or mottle symptoms. Isolate 2932 caused foliar symptoms in 16 cultivars of Cucurbita pepo, including Freedom II and Prelude II, and in six cultivars of watermelon. None of five cultivars of melon (Cucumis melo) or 11 cultivars of cucumber (Cucumis sativus) developed consistent, distinctive symptoms, but all of these cultivars were systemically infected based on back-inoculations to squash. No systemic infection of mechanically inoculated plants of 25 species representing 13 noncucurbitaceous plant families was detected. Crystalline nuclear inclusions, cytoplasmic amorphous inclusions, and cytoplasmic cylindrical inclusions were detected by light and electron microscopy in leaf tissues of infected squash and watermelon. Electron microscopy of squash leaf extracts revealed filamentous particles, and 86% of 159 particles measured ranged from 800 to 890 nm in length. The virus was transmitted in a nonpersistent manner by Myzus persicae from squash to squash in two of three trials. Immunodiffusion tests with polyclonal antisera prepared to partially purified 2932 or its capsid protein showed that the isolate was antigenically different from papaya ringspot virus type W, watermelon mosaic virus 2, and zucchini yellow mosaic virus. In limited testing of field samples of squash and watermelon since 1990, no additional isolates of the 2932 type have been found. The characteristics of isolate 2932 obtained thus far indicate that it is a distinct potyvirus. It is tentatively named watermelon leaf mottle virus to distinguish it from other potyviruses commonly isolated from cucurbits in Florida.

Occurrence of Bacterial Stem Rot, Caused by Erwinia chrysanthemi, on Field-Grown Tomato in Florida.

In September 1997, wilted 4-week-old tomato (Lycopersicon esculentum Mill.) plants were observed in a commercial production field in St. Lucie County, FL. Closer inspection of affected plants revealed hollow stems and petioles with dark, water-soaked lesions. Diseased tissue was macerated and streaked onto nutrient agar (NA) and crystal violet pectate (CVP) agar. After incubation for 2 days at 30°C, isolates produced pits on the CVP agar. Isolates were transferred onto NA and the incubation and transfer procedure was performed two additional times to obtain pure cultures. Suspensions of bacterial cells were injected into tomato and tobacco leaves to test for a hypersensitive or pathogenic reaction. Isolates produced collapsed necrotic tissue on tomato while no reaction was observed on tobacco. Tests for differentiating species and subspecies in the 'carotovora' group of Erwinia were conducted following the protocol of Dickey and Kelman (1). With known cultures of E. carotovora subsp. carotovora and E. chrysanthemi as controls, the isolate from tomato was determined to function as a facultative anaerobe, utilize asparagine as a sole source of carbon and nitrogen, and give positive reactions for pectate degradation, phosphatase, and growth at 37°C. Known cultures of E. carotovora subsp. carotovora, E. chrysanthemi, and the tomato isolate were grown on trypticase soy broth agar for 24 h at 28°C and their cellular fatty acids derivatized to fatty acid methyl esters (FAMEs). Statistical analyses of FAME profile data (MIDI Microbial Identification System, Newark, DE, version 3.60) identified the tomato isolate as Erwinia chrysanthemi. Pathogenicity was determined by inoculating 50-day-old tomato plants (cv. SunPride) with a suspension of E. chrysanthemi obtained from nutrient broth plates incubated at 24°C for 60 h. Three plants each were inoculated with the E. chrysanthemi identified from tomato, sterile water, and known cultures of E. chrysanthemi and E. carotovora subsp. carotovora by placing a drop at the junction of the petiole and stem and passing a sterile needle through the drop into the stem. Plants were maintained in a greenhouse. Dark, water-soaked cankers were observed on the stems of plants inoculated with E. chrysanthemi, including the tomato isolate and E. carotovora subsp. carotovora, after 7 days. No symptoms were observed on plants inoculated with sterile water. Reisolation of the pathogen and identification was performed with tissue from one of the symptomatic inoculated plants. Analyses of FAMEs confirmed E. chrysanthemi as the causal agent. This is the first report of E. chrysanthemi causing a vascular disease of field-grown tomato in Florida. Reference: (1) R. S. Dickey and A. Kelman. 1988. Pages 44-59 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad, ed. American Phytopathological Society, St. Paul, MN.