First Report of Colletotrichum sansevieriae Causing Anthracnose of Sansevieria trifasciata in Florida.

Sansevieria Thunberg, a member of the Agavaceae, contains around 60 species indigenous to Africa, Arabia, and India. Several species and their cultivars are commercially produced for use as interior and landscape foliage plants. During August 2010, several local nurseries submitted Sansevieria trifasciata samples to the Florida Extension Plant Diagnostic Clinic in Homestead. Leaves had round, water-soaked lesions and as the disease progressed, lesions rapidly enlarged and coalesced, resulting in severe leaf blight. Both young and mature leaves were affected. Closer examination of mature lesions revealed numerous brownish black acervuli that were produced in concentric rings, which is characteristic of anthracnose. The fungus was identified as Colletotrichum sansevieriae Nakamura based on typical cultural characteristics, conidial and appressoria morphology (1). Conidia were straight, cylindrical, obtuse at the apex, slightly acute at the base with a truncate attachment point, and 12.5 to 33 (18.4) × 4 to 8.9 (6.5) µm (n = 50). Hyphopodia were ovate, dark brown, single celled, and 6.2 to 8.7 (7.7) × 6.3 to 7.5 (7.3) µm (n = 25). Colonies on potato dextrose agar (PDA) were grayish white, felted with aerial mycelium, reverse gray to dark olivaceous gray, and partly cream in color. Sequences of the rDNA internal transcribed spacer (ITS) regions of two isolates (GenBank Accession Nos. JF911349 and JF911350) exhibited 99% nucleotide identity to an isolate of C. sansevieriae (GenBank Accession No. HQ433226) collected from diseased sansevieria in Australia. In addition, a maximum parsimony analysis (MEGA v.5.0) indicates that the two C. sansevieriae isolates from Florida are monophyletic (86% bootstrap support) with the type species from Japan (SA-1-2 AB212991; SA-1-1 AB212990) and the Australian isolate. Pathogenicity of our sequenced isolates was evaluated in greenhouse experiments. Twelve- to fourteen-week-old sansevieria plants were inoculated with conidial suspensions (1 × 106 conidia/ml) of C. sansevieriae. Inoculum or autoclaved water was sprayed over the foliage until runoff. Four plants of each of two economically important cultivars, Laurentii and Moonshine, were sprayed per treatment and the experiment was repeated twice. Inoculated plants were placed in a greenhouse at 29°C with 70 to 85% relative humidity. Plants were observed for disease development, which occurred within 10 days of inoculation for both cultivars. No symptoms developed on the control plants. Foliar lesions closely resembled those observed in the affected nurseries. C. sansevieriae was consistently reisolated from symptomatic tissue collected from greenhouse experiments. On the basis of molecular phylogenetics and distinguishing morphological characters, Nakamura et al. erected C. sansevieriae as a novel species that appears to be restricted to the host sansevieria (1). To our knowledge, this is the first report of C. sansevieriae causing anthracnose of sansevieria in Florida. Reference: (1) M. Nakamura et al. J. Gen. Plant Pathol. 72:253, 2006.

First Report of Fruit Rot on Hylocereus undatus Caused by Bipolaris cactivora in South Florida.

Pitahaya (Hylocereus undatus (Haw.) Britton & Rose), a cactus grown for its edible fruit, is gaining popularity in South Florida as part of the specialty tropical fruit market. In July 2009, flowers and fruit were discovered with an uncharacterized rot. Small, circular, light brown, depressed lesions expanded to form large areas of rot on flowers and fruit in 7 to 10 days. The lesions produced large amounts of dark fungal spores. Single-spore isolates were identified morphologically and by aligning internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) DNA sequences from the isolates with previously published sequences of Bipolaris, Drechslera, and Cochliobolus species. Conidia from the dark, blackish brown colonies were formed at the tips of pale golden brown, straight to flexuous conidiophores, 99 (184) 313 × 3 (6) 8 µm and slightly swollen at the apex and base. Conidia were pale-to-medium golden brown, smooth and clavate with a protuberant hilum, 24 (40) 51 × 9 (10) 13 µm, and two to four distoseptate. The isolates closely match descriptions of Bipolaris cactivora (= Drechslera cactivora) (3,4), although isolates from pitahaya had smaller conidia (30 to 65 µm) than previously reported. Conidial characteristics from a B. cactivora herbarium specimen BPI 431621 (U.S. National Fungus Collections) closely matched (29 (36) 50 × 8 (9) 11 µm, two to four distoseptate) our isolates. ITS (GenBank Accession Nox. HM598677-79) sequences aligned most closely (99.7% homology) with another B. cactivora isolate from China (GU390882), and both ITS and gpd (GenBank Accession Nos. HM598680-82) sequences indicate a close relationship to Bipolaris indica. Wounded or nonwounded mature pitahaya fruit and mature stems were inoculated with either a mycelia plug or a 15-µl 0.3% agar drop containing 105 conidia ml-1. Lesion diameters were measured after 7 days at 25°C, the fungus was reisolated on potato dextrose agar (PDA) and its identity was confirmed. Mean lesion diameters on mature fruit were 6.0 to 10.8 mm, depending on the inoculation method, and sporulation began 6 days after inoculation. On mature plant stems, wound-inoculated treatments formed 1.8 to 3.4 mm lesions, but nonwounded inoculations and controls were negative. Lesions were light tan, circular, and did not sporulate. To our knowledge, this is the first report of fruit rot caused by B. cactivora on pitahaya in Florida. The same pathogen causes stem rot of the Cactaceae in Europe and the United States (2) and a fruit rot on pitahaya in Japan (4). In Florida, it has been reported as causing a leaf spot on Portulaca oleracea (1). Our results indicate that B. cactivora causes flower and fruit rot on pitahaya, but does not seriously affect mature plant stems. The flower rot does not appear to significantly increase incidence but may provide inoculum for the fruit rot. The high incidence of fruit rot affecting commercial operations in Miami-Dade County over the past 2 years requires an effective disease management strategy. References: (1) S. A. Alfieri, Jr. et al. Bull. 14. Index of Plant Diseases in Florida (Revised). Florida Dep. Agric. Consumer Serv., Div. Plant Ind., 1984. (2) R. D. Durbin et al. Phytopathology 45:509, 1955. (3) M. B. Ellis. Page 432 in: Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, England. 1971. (4) S. Taba et al. J. Gen. Plant Pathol. 73:374, 2007.

First Report of Colletotrichum capsici Causing Postharvest Anthracnose on Papaya in South Florida.

Postharvest anthracnose of papaya, Carica papaya, is an important disease in most production areas worldwide (2). Colletotrichum gloeosporioides causes two types of anthracnose symptoms on papaya: (i) circular, sunken lesions with pink sporulation; and (ii) sharply defined, reddish brown and sunken lesions, described as 'chocolate spot' (2). Colletorichum spp. were isolated from lesions of the first type on papaya fruit from the University of Florida Tropical Research and Education Center, Homestead in December 2007 and from fruit imported from Belize in March 2008 (4). Single-spore isolates were identified using colony morphology and internal transcribed spacer (ITS) and mating type (MAT1-2) sequences. Two taxa were identified in both locations: (i) C. gloeosporioides (MAT1-2; GenBank Nos. GQ925065 and GQ925066) with white-to-gray, fluffy colonies with orange sporulation and straight and cylindrical conidia; and (ii) C. capsici (ITS; GenBank Nos. GU045511 to GU045514) with sparse, fluffy, white colonies with setose acervuli and falcate conidia. In addition, in Florida, a Glomerella sp. (ITS; GenBank Nos. GU045518 and GU045520 to GU045522) was recovered with darkly pigmented colonies that produced fertile perithecia after 7 to 10 days on potato dextrose agar (PDA). In each of three experiments, mature fruit (cv. Caribbean Red) were wounded with a sterile needle and inoculated with a 15-µl drop of 0.3% water agar that contained 105 conidia ml-1 of representative isolates of each taxon. The diameters of developing lesions were measured after 7 days of incubation in the dark at 25°C, and the presence of inoculated isolates was confirmed by their recovery from lesion margins on PDA. In all experiments, C. capsici and C. gloeosporioides produced lesions that were significantly larger than those that were caused by the water control and Glomerella sp. (respectively, approximately 12, 17, 0, and <1 mm in diameter). C. gloeosporioides produced sunken lesions with dark gray centers and pink/gray sporulation, which match those previously described for anthracnose on papaya (2). In contrast, C. capsici produced dark lesions due to copious setae of this pathogen; they resembled C. capsici-induced lesions on papaya that were reported previously from the Yucatan Peninsula (3). C. capsici has also been reported to cause papaya anthracnose in Asia (4), but to our knowledge, this is the first time it has been reported to cause this disease in Florida. Since it was also recovered from fruit that were imported from Belize, it probably causes anthracnose of papaya in that country as well. Another falcate-spored species, C. falcatum, was recovered from rotted papaya fruit in Texas (1). The Glomerella sp. was recovered previously from other hosts as an endophyte and causes anthracnose lesions on passionfruit (4). However, its role as a pathogen on papaya is uncertain since it was not pathogenic in the current work; the isolates that were recovered from papaya lesions may have colonized lesions that were caused by C. capsici and C. gloeosporioides. References: (1) Anonymous. Index of Plant Diseases in the United States. U.S. Dept. of Agric. Handb. No. 165. Washington, D.C., 1960. (2) D. M. Persley and R. C. Ploetz. Page 373 in: Diseases of Tropical Fruit Crops. R. C. Ploetz, ed. CABI Publishing. Wallingford, UK, 2003. (3) R. Tapia-Tussell et al. Mol Biotechnol 40:293, 2008. (4) T. L. Tarnowski. Ph.D. diss. University Florida, Gainesville, 2009.

First Report of Colletotrichum boninense, C. capsici, and a Glomerella sp. as Causes of Postharvest Anthracnose of Passion Fruit in Florida.

Anthracnose is an important foliar and fruit disease of passion fruit, Passiflora spp. (3). In 2008, postharvest anthracnose on purple and yellow passion fruits (P. edulis Sims and P. edulis f. flavicarpa O. Degner, respectively) from a commercial planting in Miami-Dade County, FL was examined. Lesions began as light brown areas that became papery, covered much of the fruit surface, and developed pink-to-dark sporulation. Single-conidium isolates from lesions were examined morphologically and with internal transcribed spacer (ITS) sequences. Four taxa were identified: Colletotrichum boninense (GenBank No. GU045516) with felted cream-to-orange colonies and cylindrical conidia; C. capsici (synonym C. truncatum [2]) (GU045515) with sparse, white mycelia, setose acervuli, and falcate conidia; C. gloeosporioides with fluffy white-to-gray colonies and straight, cylindrical conidia; and a Glomerella sp. (GU045517) with darkly pigmented perithecia. In two experiments, four mature, yellow passion fruit were wounded at a single equatorial site with a sterile needle and inoculated with a 15-µl drop of 0.3% water agar that did not contain (noninoculated control) or contained 105 conidia per ml of representative isolates from each taxon. After 21 days at 25°C without light, anthracnose incidence was recorded and the presence of the isolates was confirmed by their recovery from lesion margins on potato dextrose agar. Anthracnose did not develop on noninoculated control fruit. Mean incidences of anthracnose exceeded 50% for isolates of C. boninense (three from passion fruit), C. capsici (two from passion fruit), and a Glomerella sp. (two from passion fruit and one each from papaya and eugenia). Despite its common indictment as a causal agent of anthracnose on passion fruit (3), symptoms developed on only one fruit that was inoculated with an isolate of C. gloeosporioides from passion fruit (13%) and did not develop after inoculation with an isolate from papaya. Work is needed to determine whether host-specific populations of C. gloeosporioides exist on passion fruit that were not assessed during this study or whether the pathogen was misidentified in previous reports on this host. C. boninense was associated previously with postharvest anthracnose of passion fruit in Japan and Colombia, whereas C. capsici was associated with leaf anthracnose of passion fruit in Florida and Japan (4); both species are reported here for the first time as causes of postharvest anthracnose of passion fruit in Florida. Glomerella sp. caused darkly pigmented lesions and produced the teleomorph on symptomatic passion fruit and in single-ascospore cultures. Isolates with ITS sequences that are 99% homologous to those from passion fruit have been recovered in South Florida from eugenia, papaya, and Piper betle (4) and from other locations on several other hosts (GenBank); they are often nonpathogenic endophytes. Almeida and Coêlho (1) reported in Brazil a Glomerella sp. that formed the teleomorph in culture and caused anthracnose on passion fruit, but did not provide ITS sequences. Additional work is warranted on the identity and ecology of these fungi. References: (1) L. C. C. Almeida and R. S. B. Coêlho. Fitopatol. Bras. 32:318, 2007. (2) U. Damm et al. Fungal Divers. 39:45, 2009. (3) B. Manicom et al. Page 413 in: Diseases of Tropical Fruit Crops. R. C. Ploetz, ed. CABI Publishing, Wallingford, UK, 2003. (4) T. L. Tarnowski. Ph.D. diss. University of Florida, Gainesville, 2009.

Activity of Fungicides Against Monilinia vaccinii-corymbosi in Blueberry Flowers Treated at Different Phenological Stages.

The activity of fenbuconazole and azoxystrobin applied to blueberry flowers at different phenological stages against subsequent gynoecial infection by the mummy berry fungus Monilinia vaccinii-corymbosi was evaluated. In the greenhouse, potted blueberry plants having flower clusters at five distinct stages (from bud scale separation to anthesis) were treated with the two fungicides. One day after anthesis (between 1 and 15 days after fungicide treatment), individual flowers were detached and inoculated with conidia of M. vaccinii-corymbosi in the laboratory. Four days after inoculation, hyphal ingress into the style was determined microscopically as a measure of fungicide efficacy. Results revealed a significant flower stage effect (P < 0.0001), whereby only fungicide application at anthesis but not at the four preanthesis stages reduced subsequent fungal ingress into the style. There was no significant difference between the two fungicides (P > 0.50) nor was there a significant fungicide-flower stage interaction (P > 0.30). In the field during 2 years, mature blueberry plants were treated with the two fungicides and exposed to natural pathogen inoculum. At the time of application, flower clusters at anthesis and at three preanthesis stages were selected and tagged. Mummy berry incidence in fruit developing from the tagged clusters was assessed to determine treatment effects. Whereas fenbuconazole lowered disease incidence for all preanthesis stages, azoxystrobin was effective only at the latest preanthesis stage. The discrepancy between these results and those of the greenhouse study (where there was no preanthesis activity of either fungicide) indirectly suggests post-infection fungicidal activity in the ovary, the base of which was exposed to the fungicide spray at the time of treatment for all flower phenology stages. Thus, although there appears to be insufficient translocation of the two fungicides in flowers treated at preanthesis stages to prevent stylar ingress by the pathogen, fungicidal activity in the ovary may be sufficient to halt subsequent fungal colonization, especially for fenbuconazole. To prescribe the most effective management program for flower-infecting fungi, translocation and post-infection activity of fungicides in floral tissues must be better understood.

Effect of high-dose oral ganciclovir on didanosine disposition in human immunodeficiency virus (HIV)-positive patients.

This study was designed to investigate the interaction between high-dose oral ganciclovir (6,000 mg/day) and didanosine at steady state in patients who were seropositive for human immunodeficiency virus (HIV) and cytomegalovirus (CMV) infection. The study was conducted as an open-label, randomized, three-period crossover study. Patients received (in random order) multiple oral doses of didanosine 200 mg every 12 hours alone, ganciclovir 2,000 mg every 8 hours alone, and ganciclovir 2,000 mg every 8 hours in combination with didanosine 200 mg every 12 hours. Blood and urine samples for determinations of drug concentrations were obtained on day 3 of each dose regimen. When ganciclovir was administered either before or 2 hours after didanosine, the mean increases in maximum concentration (Cmax), area under the concentration-time curve (AUC0-12), and percent excreted in urine of didanosine were 58.6% and 87.3%, 87.3% and 124%, and 100% and 153%, respectively. There were no statistically significant effects of didanosine on the steady-state pharmacokinetics of ganciclovir in the presence of didanosine, irrespective of sequence of administration. There were no significant changes in renal clearance of didanosine, suggesting that the mechanism for the interaction does not involve competition for active renal tubular secretion. The mechanism responsible for increased didanosine concentrations and percent excreted in urine during concurrent ganciclovir therapy may be a result of increased bioavailability of didanosine. However, the mechanism appears to be saturated at oral ganciclovir doses of 3 g/day.

Pharmacokinetics of oral ganciclovir alone and in combination with zidovudine, didanosine, and probenecid in HIV-infected subjects.

The aim of this study was to determine whether oral ganciclovir interacted pharmacokinetically with zidovudine (AZT), didanosine (ddI), or probenecid. A multicenter, open-label, randomized, crossover pharmacokinetic study with four phases was undertaken at an outpatient private research center and at university research clinics. Twenty-six HIV-infected adults (23 men, 3 women) with cytomegalovirus (CMV) seropositivity and CD4+ T-lymphocyte count > or =100 cells/microl were studied. Patients had to be stable on antiretroviral therapy for at least 4 weeks. Patients with a history of opportunistic infection or gastrointestinal symptoms were excluded. Measurements included serial blood and urine samples during the dosing intervals at steady state. The steady-state pharmacokinetics of ganciclovir were determined after the participants had stabilized and were tolerating AZT or ddI therapy. When a 1000-mg dose of oral ganciclovir was taken every 8 hours, there was a significant mean increase in Cmax and dosing interval area under the serum concentration time curve over a dosing interval (AUC) for the two antiretroviral drugs: for AZT, 61.6% and 19.5%, respectively; for ddI when administered sequentially (2 hours before ganciclovir), 116.0% and 114.6%; and for ddI administered simultaneously with ganciclovir, 107.9% and 107.1%, respectively. There was no significant change in renal clearance for either antiretroviral drug, suggesting that the interaction did not occur through a renal mechanism. There was no significant change in mean ganciclovir Cmax and AUC(0-8) when coadministered with AZT. Mean increases in Cmax and AUC(0-8) of oral ganciclovir averaged 40.1% and 52.5%, respectively, when coadministered with probenecid, but decreased by 22.1% and 22.7%, respectively, when oral ganciclovir was administered 2 hours after ddI. There was no change in the mean ganciclovir Cmax or AUC(0-8) when administered simultaneously with ddI. The mean renal clearance of oral ganciclovir was not affected by AZT or ddI coadministration intake, but there was a mean decrease of 19% when coadministered with probenecid. We conclude the increased serum concentration and reduced renal clearance of ganciclovir suggests competition with probenecid for secretion at the renal tubule. The mechanism of the interaction of oral ganciclovir with either AZT or ddI remains to be determined. The magnitude of the effect of oral ganciclovir on ddI pharmacokinetics may result in an increase in ddI concentration-related toxicities. Similarly, the small but significant decrease in ganciclovir concentration with sequential combination ddl therapy may impair the efficacy of oral ganciclovir. For HIV-infected patients receiving ganciclovir and ddI, clinicians should recommend administering the two drugs simultaneously, and patients should be monitored closely for ddI-associated toxicities.

Simultaneous determination of mycophenolic acid and its glucuronide conjugate in human plasma by a single-run ion-paring method.

A quick and robust HPLC method for simultaneous determination of plasma concentrations of mycophenolic acid (MPA) and its glucuronide conjugate (MPAG) is described. Using tetrabutylammonium dihydrogen phosphate as ion-paring reagent in the mobile phase, concentrations for both MPA and MPAG can be determined in a single HPLC run on a reversed-phase C18 column at 254 nm. The method is reproducible and accurate, with lower limits of quantification of 0.100 microg/ml for MPA and 0.800 microg/ml for MPAG. The quantification ranges of the method are 0.100-40.0 microg/ml for MPA and 0.800-400 microg/ml for MPAG using a 0.5-ml aliquot in the analysis.