Teresa E. Seijo

New report of Colletotrichum gloeosporioides causing postbloom fruit drop on citrus in Bermuda

Abstract Postbloom fruit drop (PFD) of citrus was observed for the first time after a widespread and severe outbreak occurred in Bermuda in the 1990s. Fruit losses from the disease were estimated at 25% to 35% in sweet orange, grapefruit, lemon and Tahiti lime. The causal agent of PFD has been reported as either Colletotrichum gloeosporioides or Colletotrichum acutatum. Bermuda isolates of Colletotrichum recovered from diseased orange trees produced lesions typical of PFD in detached petals of orange, lime and grapefruit, and in attached orange blossoms. The isolates produced few to no lesions of anthracnose on leaves of key lime seedlings. Kochs postulates were fulfilled following reisolation of morphologically identical fungi from inoculated tissues. The isolates were characterized using morphological (conidial size and shape and colony colour), physiological (growth rate at 24 °C and on a benomyl-amended medium), immunological (ELISA) and molecular (PCR amplification and sequencing of the ITS region) methods. Immunological and molecular techniques provided definitive identification of the isolates as Colletotrichum gloeosporioides. This report confirms that C. gloeosporioides can be the causal agent of PFD; therefore, identification of the causal agent to species by immunology, molecular analysis and fungicide sensitivity is suggested for new outbreaks of the disease.

First Report of Macrophomina phaseolina Causing a Crown Rot of Strawberry in Florida

Strawberry (Fragaria × ananassa Duchesne) is produced as an annual winter crop in raised, plastic-mulched beds on 2,800 ha in west central Florida. In December 2001, a grower submitted collapsed and dying strawberry plants from a commercial field to the University of Florida in Dover. The cut crowns of affected plants revealed dark brown necrotic areas on the margins and along the woody vascular ring. Macrophomina phaseolina was isolated from pieces of infected tissue cut aseptically from the crowns and placed on a medium containing 12 g of Difco potato dextrose broth, 17 g of Bacto agar, 250 mg of ampicillin, and 100 mg of streptomycin sulfate per liter of water. The fungus produced numerous, dark, oblong sclerotia in the isolation medium after 4 to 5 days incubation at 24°C under constant fluorescent lighting. In 10-day-old cultures, sclerotia ranged in size from 55 to 190 μm long by 50 to 135 μm wide (average 105 × 74 μm). Ostiolate pycnidia bearing relatively large, broadly ellipsoidal, hyaline conidia occasionally developed on the host tissue after 8 to 10 days of incubation (2). During the 2003-2004 season, M. phaseolina was isolated from dying strawberry plants taken from the original field and two additional farms. Affected plants were often found along field margins or other areas inadequately fumigated with methyl bromide. Two single-spore isolates from different fields were tested for pathogenicity on nursery runner plants (cv. Strawberry Festival) grown for 4 weeks in the greenhouse on artificial potting soil. The fungal isolates were grown on corn meal agar at 24°C for 4 days and allowed to colonize sterile wooden toothpicks placed on the medium for an additional 5 days. Prior to use, the toothpicks were sterilized by autoclaving twice in deionized water and a third time in V8 juice. Six plants were inoculated with each isolate by inserting a colonized toothpick into each crown. Sterile, V8-infused toothpicks were inserted into the crowns of corresponding control plants. The plants were incubated in a greenhouse in a randomized complete block design with two replicates of three plants each. After 3 days, 33 to 100% of the inoculated plants developed wilting in one or more leaves. All inoculated plants collapsed or died within 2 weeks of inoculation, while the control plants remained healthy during the observation period. The pathogen was readily reisolated from inoculated plants. Charcoal rot disease caused by M. phaseolina has been reported on strawberry in France, India, and Illinois (2,3). To our knowledge, this is the first report from Florida. M. phaseolina may be an emerging threat as the Florida strawberry industry transitions from methyl bromide to other fumigants in 2005. References: (1) J. Maas. Macrophomina leaf blight and dry crown rot. Page 26 in: Compendium of Strawberry Diseases. 2nd ed. J. L. Maas, ed. The American Phytopathological Society, St. Paul, MN, 1998. (2) G. S. Smith and T. D. Wyllie. Charcoal rot. Pages 29-31 in: Compendium of Soybean Diseases. G. L. Hartman et al., eds. 4th ed. The American Phytopathological Society, St. Paul, MN. 1999. (3) B. Tweedy et al. Plant Dis. Rep. 42:107, 1958.

Colletotrichum fragariae Is a Pathogen on Hosts Other Than Strawberry

Evidence that Colletotrichum fragariae causes disease on hosts other than strawberry is limited. In the fall of 2006, fungal isolates from silver date palm with leaf spot symptoms and from cyclamen with leaf spot and stem rot symptoms were identified as C. fragariae. After confirming the pathogenicity of the isolates on their host of origin, a representative isolate from each host was compared to C. fragariae and to C. gloeosporioides/Glomerella cingulata isolates from strawberry. Date palm and cyclamen isolates bore conidia on setae, and conidia were tapered and in the size range of C. fragariae reference isolates. Sequence data from the combined internal transcribed spacer (ITS) regions 1 and 2 and the gene for the 5.8 ribosomal RNA from the cyclamen and date palm isolates matched the sequence for C. fragariae reference isolates. Based on these characteristics, it was concluded that the C. fragariae species designation was correct for both isolates. However, the date palm isolate was a weak pathogen on strawberry compared with other isolates and had a distinct AT-rich DNA banding pattern. The ability of the cyclamen isolate to cause crown rot on strawberry was comparable with the strawberry reference isolates, and the AT-rich DNA banding pattern of the cyclamen isolate was identical to the C. fragariae isolates from strawberry. The results indicate that C. fragariae is a pathogen on hosts other than strawberry and that there is more diversity among C. fragariae isolates than previously reported.

Resistance in Strawberry Isolates of Colletotrichum acutatum from Florida to Quinone-Outside Inhibitor Fungicides

Anthracnose fruit rot of strawberry, caused by Colletotrichum acutatum, is a major disease in Florida and frequent quinone-outside inhibitor (QoI) fungicide applications are needed for disease control. From 1994 to 2014, 181 C. acutatum isolates were collected from multiple strawberry fields in Florida with or without QoI spray history. Sensitivity to azoxystrobin and pyraclostrobin was tested based upon mycelial growth and germ tube elongation inhibition. Mean effective concentration where growth was reduced by 50% (EC50) values for isolates collected prior to 2013 based upon mycelial growth were 0.22 and 0.013 μg/ml and upon germ tube elongation were 0.57 and 0.03 μg/ml for azoxystrobin and pyraclostrobin, respectively. Mycelial growth and germ tube elongation of 48 isolates collected in 2013 and 2014 were not inhibited with azoxystrobin at 3 μg/ml and pyraclostrobin at 0.110 μg/ml. A fungicide discriminatory dose assay indicated that 43 of the 48 isolates had EC50 values higher than 100 and 10 μg/ml for azoxystrobin and pyraclostrobin, respectively. Azoxystrobin and pyraclostrobin sprayed preventively on strawberry fruit inoculated with C. acutatum failed to control resistant isolates. Sequencing of the cytochrome b gene of sensitive and resistant isolates showed that QoI-resistant isolates contained either G143A or F129L amino acid substitutions.

Evaluating Weeds as Hosts of Tomato yellow leaf curl virus

ABSTRACT Bemisia tabaci (Gennadius) biotype B transmits Tomato yellow leaf curl virus (TYLCV), which affects tomato production globally. Prompt destruction of virus reservoirs is a key component of virus management. Identification of weed hosts of TYLCV will be useful for reducing such reservoirs. The status of weeds as alternate hosts of TYLCV in Florida remains unclear. In greenhouse studies, B. tabaci adults from a colony reared on TYLCV-infected tomato were established in cages containing one of four weeds common to horticultural fields in central and south Florida. Cages containing tomato and cotton were also infested with viruliferous whiteflies as a positive control and negative control, respectively. Whitefly adults and plant tissue were tested periodically over 10 wk for the presence of TYLCV using PCR. After 10 wk, virus-susceptible tomato plants were placed in each cage to determine if whiteflies descended from the original adults were still infective. Results indicate that Bidens alba, Emilia fosbergii, and Raphanus raphanistrum are not hosts of TYLCV, and that Amaranthus retroflexus is a host.

First report of Acidovorax avenae subsp. avenae causing bacterial leaf stripe of Strelitzia nicolai.

White bird of paradise (Strelitzia nicolai Regel & K. Koch) is a commonly grown ornamental in central and south Florida. Each summer of 2004 to 2007, a reoccurring disease was observed at a commercial nursery in central Florida. Diseased plants had brown, necrotic stripes between the lateral leaf veins, which usually appeared along the midvein and spread toward the leaf edge. Lesions developed on the youngest leaves as they emerged from the central whorl. During 2004 and 2005, 20 symptomatic leaves were sampled. A white, nonfluorescent bacterium was consistently isolated from symptomatic tissue. It induced a hypersensitive response (HR) on tomato, grew at 41°C, and was identified as a Acidovorax sp. based on fatty acid analysis and as Acidovorax avenae subsp. avenae by Biolog metabolic phenotype analysis (similarity 0.76 to 0.86). A partial 16S rRNA gene sequence (1,455 bp) (Accession No. EF418616) was identical to four sequences in the NCBI (National Center for Biotechnology Information) database: one from A. avenae subsp. avenae and three from A. avenae of undetermined subspecies. To confirm pathogenicity, a bacterial suspension (O.D590 = 0.1) was applied to fill the central whorl (~0.5 to 1 ml) of potted S. nicolai. Plants were incubated for 7 to 10 days inside plastic bags at ambient temperature. Plants were inoculated individually with five strains of A. avenae subsp. avenae, four from S. nicolai, and one from corn (ATCC19860). Two to nine plants per strain were inoculated in each experiment. All strains were tested at least twice and noninoculated control plants were included. Symptoms were reproduced on the emerging leaf of 50 to 100% of inoculated plants with all five A. avenae subsp. avenae strains. No symptoms were observed on the controls. The bacteria recovered from symptomatic tissue were confirmed to be A. avenae subsp. avenae. Corn seedlings were inoculated as described above, except that entire seedlings were sprayed. Water-soaked lesions along the length of older leaf blades developed in 4 to 7 days. Only the corn strain was pathogenic (>80% of seedlings symptomatic), indicating host specificity. To our knowledge, this is the first report of A. avenae subsp. avenae infecting S. nicolai. In 1971, Wehlburg (2) described the same symptoms on orange bird of paradise (S. reginae) as being caused by a nonfluorescent Pseudomonas sp. This report likely describes the same disease since the published description is consistent with symptoms caused by A. avenae subsp. avenae. The pathogen reported by Wehlburg (2) had one polar flagellum, reduced nitrate, produced oxidase and a HR, and utilized arabinose, but not sucrose or arginine, characteristics consistent with those of A. avenae subsp. avenae (1). The only difference was A. avenae subsp. avenae has a delayed positive starch hydrolysis (1), whereas Welhburgs strain was negative. This disease occurs mainly on young leaves when plants receive daily overhead irrigation. Incidence can be as high as 40%, occasionally causing mortality, but even mild symptoms affect appearance and reduce marketability as an ornamental. References: (1) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001. (2) C. Wehlburg. Plant Dis. Rep. 55:447, 1971.