First Look Into the Ambrosia Beetle-Fungus Symbiosis Present in Commercial Avocado Orchards in Michoacán, Mexico.

Most beetle-fungus symbioses do not represent a threat to agricultural and natural ecosystems; however, a few beetles are able to inoculate healthy hosts with disease-causing fungal symbionts. Here, we report the putative nutritional symbionts associated with five native species of ambrosia beetles colonizing commercial avocado trees in four locations in Michoacán. Knowing which beetles are present in the commercial orchards and the surrounding areas, as well as their fungal associates, is imperative for developing a realistic risk assessment and an effective monitoring system that allows for timely management actions. Phylogenetic analysis revealed five potentially new, previously undescribed species of Raffaelea, and three known species (R. arxi, R. brunnea, R. fusca). The genus Raffaelea was recovered from all the beetle species and across the different locations. Raffaelea lauricola (RL), which causes a deadly vascular fungal disease known as laurel wilt (LW) in Lauraceae species, including avocado, was not recovered. This study points to the imminent danger of native ambrosia beetles spreading RL if the pathogen is introduced to Mexico's avocado orchards or natural areas given that these beetles are associated with Raffaelea species and that lateral transfer of RL among ambrosia beetles in Florida suggests that the likelihood of this phenomenon increases when partners are phylogenetically close. Therefore, this study provides important information about the potential vectors of RL in Mexico and other avocado producing regions. Confirming beetle-fungal identities in these areas is especially important given the serious threat laurel wilt disease represents to the avocado industry in Mexico.

In-Grove Spatiotemporal Spread of Citrus Huanglongbing and Its Psyllid Vector in Relation to Weather.

Reports of spatial patterns of 'Candidatus Liberibacter asiaticus'-infected asymptomatic citrus trees and 'Ca. L. asiaticus'-positive Asian citrus psyllids (ACP) are rare, as are published relationships between huanglongbing (HLB), ACP, and weather. Here, spatial patterns of 'Ca. L. asiaticus'-positive asymptomatic and symptomatic trees were determined every half year in a small grove over 2.5 years, and of HLB-symptomatic trees and ('Ca. L. asiaticus'-positive) ACP populations every month in two commercial groves for 1 year. Spread of symptomatic trees followed that of asymptomatic 'Ca. L. asiaticus'-positive trees with <6 months' delay. 'Ca. L. asiaticus'-positive asymptomatic and symptomatic fronts moved at 2.5 to 3.6 m month-1. No spatial relationship was detected between ACP populations and HLB-infected trees. HLB incidence and 'Ca. L. asiaticus'-positive ACP dynamics were tentatively positively correlated with monthly rainfall data and, to a lesser extent, with average minimum temperature.

A Novel Disease of Big-Leaf Mahogany Caused by Two Fusarium Species in Mexico.

Big-leaf mahogany (Swietenia macrophylla) is valued for its high-quality wood and use in urban landscapes in Mexico. During surveys of mango-producing areas in the central western region of Mexico, symptoms of malformation, the most important disease of mango in the area, were observed on big-leaf mahogany trees. The objectives of this research were to describe this new disease and determine its cause. Symptoms on big-leaf mahogany at four sites in Michoacán, Mexico resembled those of the vegetative phase of mango malformation, including compact, bunched growth of apical and lateral buds, with greatly shortened internodes and small leaves that curved back toward the supporting stem. Of 163 isolates that were recovered from symptomatic tissues, most were identified as Fusarium pseudocircinatum (n = 121) and F. mexicanum (n = 39) using molecular systematic data; two isolates represented unnamed phylospecies within the F. incarnatum-equiseti species complex (FIESC 20-d and FIESC 37-a) and another was in the F. solani species complex (FSSC 25-m). However, only F. mexicanum and F. pseudocircinatum induced malformation symptoms on 14-day-old seedlings of big-leaf mahogany. The results indicate that F. mexicanum and F. pseudocircinatum, previously reported in Mexico as causal agents of mango malformation disease, also affect big-leaf mahogany. This is the first report of this new disease and the first time that F. mexicanum was shown to affect a host other than mango.

Regional Spatial-Temporal Spread of Citrus Huanglongbing Is Affected by Rain in Florida.

Citrus huanglongbing (HLB), associated with 'Candidatus Liberibacter asiaticus' (Las), disseminated by Asian citrus psyllid (ACP), has devastated citrus in Florida since 2005. Data on HLB occurrence were stored in databases (2005 to 2012). Cumulative HLB-positive citrus blocks were subjected to kernel density analysis and kriging. Relative disease incidence per county was calculated by dividing HLB numbers by relative tree numbers and maximum incidence. Spatiotemporal HLB distributions were correlated with weather. Relative HLB incidence correlated positively with rainfall. The focus expansion rate was 1626 m month-1, similar to that in Brazil. Relative HLB incidence in counties with primarily large groves increased at a lower rate (0.24 year-1) than in counties with smaller groves in hotspot areas (0.67 year-1), confirming reports that large-scale HLB management may slow epidemic progress.

Comparing Avocado, Swamp Bay, and Camphortree as Hosts of Raffaelea lauricola Using a Green Fluorescent Protein (GFP)-Labeled Strain of the Pathogen.

Raffaelea lauricola, a fungal symbiont of the ambrosia beetle Xyleborus glabratus, causes laurel wilt in members of the Lauraceae plant family. North American species in the family, such as avocado (Persea americana) and swamp bay (P. palustris), are particularly susceptible to laurel wilt, whereas the Asian camphortree (Cinnamomum camphora) is relatively tolerant. To determine whether susceptibility is related to pathogen colonization, a green fluorescent protein-labeled strain of R. lauricola was generated and used to inoculate avocado, swamp bay, and camphortree. Trees were harvested 3, 10, and 30 days after inoculation (DAI), and disease severity was rated on a 1-to-10 scale. By 30 DAI, avocado and swamp bay developed significantly more severe disease than camphortree (mean severities of 6.8 and 5.5 versus 1.6, P < 0.003). The extent of xylem colonization was recorded as the percentage of lumena that were colonized by the pathogen. More xylem was colonized in avocado than camphortree (0.9% versus 0.1%, P < 0.03) but colonization in swamp bay (0.4%) did not differ significantly from either host. Although there were significant correlations between xylem colonization and laurel wilt severity in avocado (r = 0.74), swamp bay (r = 0.82), and camphortree (r = 0.87), even severely affected trees of all species were scarcely colonized by the pathogen.

First Report of Mango Malformation Disease Caused by Fusarium pseudocircinatum in Mexico.

Mango (Mangifera indica L.) malformation disease (MMD) is one of the most important diseases affecting this crop worldwide, causing severe economic loss due to reduction of yield. After the first report in India in 1891 (3), MMD has spread worldwide to most mango-growing regions. Several species of Fusarium cause the disease, including F. mangiferae in India, Israel, the USA (Florida), Egypt, South Africa, Oman, and elsewhere; F. sterilihyphosum in South Africa and Brazil; F. proliferatum in China; F. mexicanum in Mexico; and recently, F. tupiense in Brazil (1,2,3,4). Besides F. mexicanum, F. pseudocircinatum, not yet reported as a causal agent of MMD, was isolated in Mexico from affected inflorescences and vegetative malformed tissues (4). Symptoms of vegetative malformation caused by F. pseudocircinatum included hypertrophied, tightly bunched young shoots, with swollen apical and lateral buds producing misshapen terminals with shortened internodes and dwarfed leaves. Infected inflorescences of primary or secondary axes on affected panicles were shortened, thickened, and highly branched, while the peduncles became thick, remained green and fleshy, and branches profusely resembled a cauliflower in shape and size (3). Ten isolates of F. pseudocircinatum were recovered from cultivars Ataulfo, Criollo, Haden, and Tommy Atkins in Guerrero, Campeche, and Chiapas states and characterized. Isolates produced mostly 0-septate but occasionally 1- to 3-septate oval, obovoid, or elliptical aerial conidia (0-septate: 4 to 19 [avg. 8.7] × 1.5 to 4 [avg. 2.6] µm) in false heads in the dark and in short false chains under black light, unbranched or sympodially branched prostrate aerial conidiophores producing mono- and polyphialides, and sporodochia with straight or falcate conidia that were mostly 3- to 5-septate, but sometimes up to 7-septate (3-septate: 25 to 58 [avg. 41] × 2 to 3.3 [avg. 2.9] µm; 5-septate: 33.5 to 76.5 [avg. 56.7] × 2.5 to 6 [avg. 3.5] µm). Circinate sterile hyphae were rarely formed. Two representative isolates, NRRL 53570 and 53573, were subjected to multilocus molecular phylogenetic analyses of portions of five genes: nuclear large subunit 28S ribosomal RNA, ß-tubulin, calmodulin, histone H3, and translation elongation factor (TEF)-1α (GenBank GU737456, GU737457, GU737290, GU737291, GU737371, GU737372, GU737425, GU737426, GU737398, and GU737399). Two pathogenicity tests were conducted with NRRL 53570 and 53573 on healthy 2-year-old nucellar seedlings of polyembryonic Criollo; 20 µl conidial suspensions (5 × 106 conidia/ml) of each isolate and water controls were inoculated separately on 15 buds on 3 different trees, as described previously (1). The following conditions were used in experiment 1: 24 to 27°C with light intensity of 16.2 to 19.8 •Mol m-2s-1 in the range of 400 to 700 nm, and photoperiods of 14 h light and 10 h dark. Typical vegetative disease symptoms were discernible in plants inoculated with NRRL 53570 (20%) and 53573 (7%) after 8 months. In experiment 2, after 3 months growth under the above conditions, seedlings were transferred to an outdoor nursery in Iguala, Guerrero. Typical vegetative symptoms of MMD were observed in 86.7 and 13.3% of the buds inoculated with F. pseudocircinatum NRRL 53570 and 53573, respectively, after 9 months. Isolates from typical symptomatic vegetative buds were confirmed as F. pseudocircinatum by sequencing a portion of their TEF-1α gene, thus fulfilling Koch's postulates. This is the first report of F. pseudocircinatum as a causal agent of MMD. References: (1) S. Freeman et al. Phytopathology 89:456, 1999. (2) C. S. Lima et al. Mycologia 104:1408, 2012. (3) W. F. O. Marasas et al. Phytopathology 96:667, 2006. (4) G. Otero-Colina et al. Phytopathology 100:1176, 2010.

First Report of Fusarium oxysporum f. sp. cubense Tropical Race 4 Associated with Panama Disease of Banana outside Southeast Asia.

Fusarium wilt or Panama disease of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive plant diseases (3). Race 1 ravaged 'Gros Michel'-based export trades until the cultivar was replaced by resistant Cavendish cultivars. However, a new variant of Foc, tropical race 4 (TR4), was identified in Southeast Asia in 1992 and has spread throughout the region (3). Cavendish clones, which are most important in subsistence and export production, are among the wide range of cultivars that are affected, and there is a huge concern that TR4 will further disseminate in Africa since its presence was announced in November 2013 and move into Latin America, thereby threatening other vital banana-growing regions. In Jordan, Cavendish bananas are produced on 1,000 to 1,500 ha in the Jordan Valley (32°N, 35.5°E). In 2006, symptoms of Fusarium wilt were observed and sampled for the isolation of Foc. On half-strength PDA amended with 100-ppm streptomycin sulfate, pale salmon-colored colonies with floccose mycelia developed consistently from surface-disinfested xylem. Single microconidia from these colonies were transferred to half-strength PDA, and conidia and mycelia from these monospore colonies were stored at -80°C in 15% glycerol. On banana leaf agar (Co60-irradiated leaf tissue on water agar), isolates resembled F. oxysporum phenotypically by producing infrequent three- to five-celled macroconidia, copious, usually aseptate microconida on monophialides, and terminal and intercalary chlamydospores after 2 weeks (2). With nitrate-nonutilizing (nit) mutants and testers for different vegetative compatibility groups (VCGs), each of seven examined monospore isolates were placed in VCG 01213, which contains only strains of TR4 (3). Total DNA was extracted from six isolates and PCR analyses, which confirmed their identity as TR4 (1). Subsequently, one of the isolates (JV11) was analyzed for pathogenicity. Inoculum production and inoculation were according to (1) by dipping (30 min) root-wounded 10-week-old plants of the Cavendish cv. Grand Naine in 2 liters of spore suspension (1.0 × 106 spores/ml). Inoculated plants were then placed in sand in 3-liter pots under 28°C, 70% relative humidity, and a 16/8-h light/darkness photoperiod. Sets of three plants were each treated with either JV11 or two TR4 controls (isolate II-5 and a strain isolated from an affected Cavendish plant in Mindanao, Philippines, both of which were diagnosed as TR4 by PCR and pathogenicity analyses). Control sets were either treated with race 1 originating from Cruz das Almas, Bahia, Brazil (1), or water. After 2 weeks, plants inoculated with JV11 and TR4 controls produced typical symptoms of Fusarium wilt. After 4 weeks, tissue was collected from all plants and plated on Komada's medium. TR4 was directly confirmed by PCR (1), either directly from symptomatic plants (JV11 and TR4 controls), or from isolates that were recovered from these plants. Nothing was re-isolated from race 1 inoculated plants and water controls, which remained asymptomatic. This is the first report of TR4 affecting Cavendish outside Southeast Asia, is its northernmost outbreak, and represents a dangerous expansion of this destructive race. Currently, 80% of the Jordan Valley production area is affected by Fusarium wilt, and 20 to 80% of the plants are affected in different farms. References: (1) M. A. Dita et al. Plant Pathol. 59:348, 2010. (2) J. F. Leslie and B. A. Summerell. The Fusarium Lab Manual. Blackwell, Ames, 2006. (3) R. C. Ploetz. Phytopathology 96:653, 2006.

First Report of Rust Caused by Puccinia nakanishikii on Lemongrass, Cymbopogon citratus, in Florida.

Lemongrass, Cymbopogon citratus (DC.) Stapf. (Poaceae), is grown widely in the tropics and subtropics as an ornamental, flavoring ingredient in Asian cooking, and for tea and fragrant oil (3). In February 2013, rust symptoms were observed on lemongrass in several gardens in Miami-Dade County, Florida. Symptoms began as small chlorotic flecks on both leaf surfaces that became crimson and enlarged to streaks ~1 cm in length. On the abaxial side of leaves, erumpent streaks ruptured to produce pustules in which urediniospores formed. Eventually, streaks coalesced to produce large patches of tan to purplish necrotic tissue that blighted most of the leaf surface and was often surrounded by chlorotic borders. These symptoms, fungal morphology, and nuclear ribosomal large subunit (28S) DNA analysis were used to identify the pathogen as Puccinia nakanishikii Dietel. Urediniospores were pyriform to globose, orange to crimson, slightly echinulate, and somewhat longer than a previous report (32.1 ± 3.4 (27 to 42) × 23.3 ± 2.4 (21 to 27) µm vs. 22 to 28 × 22 to 25 µm) (2). Uredinia contained clavate paraphyses, but teliospores were not observed. No aecial host is known for this pathogen. A 28S DNA sequence that was generated with the NL1 and LR3 primers (1,4) was deposited in GenBank under accession no. KC990123; it shared 99% identity with GenBank accession GU058002, which came from a specimen of P. nakanishikii in Hawaii. Voucher specimens of affected leaves of lemongrass have been deposited at the Arthur Herbarium, Purdue University. Although this disease has been reported in California, Hawaii, New Zealand, and Thailand, this is believed to be the first report from Florida (2). Based on rainfall and temperature conditions that are conducive to its development in South Florida, it has the potential to significantly reduce the health and production of this plant in area gardens. References: (1) C. P. Kurtzman and C. J. Robnett. Antonie Van Leeuwenhoek 73:331. 1998. (2) S. Nelson. Rust of Lemongrass. Univ. Hawaii PD-57, 2008. (3) USDA, ARS, GRIN Online Database. URL: http://www.ars-grin.gov/cgi-bin/npgs/html/taxon.pl?12797 , accessed 25 April 2013. (4) R. Vilgalys and M. Hester. J Bacteriol. 172:4238, 1990.

First Report of Gulf Licaria, Licaria trianda, as a Suscept of Laurel Wilt.

Gulf licaria, Licaria trianda (Sw.) Kosterm., is a federally endangered member of the Lauraceae plant family in Miami-Dade County, Florida. It was never common in the area, and urban development has extirpated it from most of its former range; as of 2001, fewer than 10 trees remained in a single, remnant habitat in the continental United States, Simpson Park (25°45'31″N, 80°11'46″W) (2). Laurel wilt, caused by the fungus Raffaelea lauricola T. C. Harr., Fraedrich & Aghayeva, has recently devastated members of the Lauraceae in the southeastern United States, most notably redbay, Persea borbonia (1). As R. lauricola and its vector, the redbay ambrosia beetle Xyleborus glabratus, have spread in the region, an increasing number of taxa in this plant family have been affected by this disease (1). In 2012, seedlings of gulf licaria and redbay were obtained from local nurseries; they were grown in 30 liter pots, 1.3 m tall, had stems 3 cm in diameter 20 cm above the soil line, and were maintained with standard watering and fertilization practices. In two pathogenicity experiments on July 6 and September 25, 2012, three plants each of gulf licaria and redbay were inoculated with an isolate of R. lauricola, RL4, as described in previous experiments (3), and two plants each were mock inoculated (water control). RL4 is deposited as CBS 127349 at the Centraalbureau voor Schimmelcultures (CBS Fungal Biodiversity Centre, Utrecht, The Netherlands), and a SSU rDNA sequence for it is deposited in GenBank under Accession No. HM446155. Beginning 2 weeks after inoculation, plants were rated on a weekly basis for the development of external symptoms, on a subjective 1 (no symptoms) to 10 (dead) scale (3). After 5 weeks, inoculated plants of redbay in each experiment (positive control) had died after first developing symptoms of wilt and necrotic foliage that are typical for this disease (1). In contrast, inoculated plants of gulf licaria developed severe symptoms by the time experiments were terminated 6 and 11 weeks after inoculation; chlorosis developed on some of the leaves of all plants and these eventually abscised (mean external severities of 7.3 and 6.5, respectively), but plants did not die. Brown to greyish discoloration of sapwood developed in all inoculated plants, and the pathogen was recovered from symptomatic sapwood on CSMA (3). No symptoms developed on mock inoculated plants and the pathogen was not recovered from them. It is concluded that gulf licaria is susceptible to laurel wilt, but that it is apparently less susceptible than redbay. Whether X. glabratus is attracted to, or will bore into, gulf licaria is not known, but will play a significant role in the extent to which this rare tree is affected by laurel wilt. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) G. D. Gann et al. Rare Plants of South Florida: Their History, Conservation, and Restoration. Institute for Regional Conservation, Miami, 2002. (3) R. C. Ploetz et al. Plant Pathol. 61:801, 2012.

Current status of the taxonomic position of Fusarium oxysporum formae specialis cubense within the Fusarium oxysporum complex.

Fusarium oxysporum is an asexual fungal species that includes human and animal pathogens and a diverse range of nonpathogens. Pathogenic and nonpathogenic strains of this species can be distinguished from each other with pathogenicity tests, but not with morphological analysis or sexual compatibility studies. Substantial genetic diversity among isolates has led to the realization that F. oxysporum represents a complex of cryptic species. F. oxysporum f. sp cubense (Foc), causal agent of Fusarium wilt of banana, is one of the more than 150 plant pathogenic forms of F. oxysporum. Multi-gene phylogenetic studies of Foc revealed at least eight phylogenetic lineages, a finding that was supported by random amplified polymorphic DNAs, restriction fragment length polymorphisms and amplified fragment length polymorphisms. Most of these lineages consist of isolates in closely related vegetative compatibility groups, some of which possess opposite mating type alleles, MAT-1 and MAT-2; thus, the evolutionary history of this fungus may have included recent sexual reproduction. The ability to cause disease on all or some of the current race differential cultivars has evolved convergently in the taxon, as members of some races appear in different phylogenetic lineages. Therefore, various factors including co-evolution the plant host and horizontal gene transfer are thought to have shaped the evolutionary history of Foc. This review discusses the evolution of Foc as a model formae specialis in F. oxysporum in relation to recent research findings involving DNA-based studies.

Laurel Wilt, Caused by Raffaelea lauricola, is Confirmed in Miami-Dade County, Center of Florida's Commercial Avocado Production.

Laurel wilt, caused by Raffaelea lauricola, threatens native and nonnative species in the Lauraceae in the southeastern United States, including the important commercial crop, avocado, Persea americana (2,4). Although the pathogen's vector, Xyleborus glabratus, was detected in Miami-Dade County, FL in January 2010, laurel wilt had not been reported (4). In February 2011, symptoms of the disease were observed on native swampbay, P. palustris, in Miami-Dade County (25°72'N, 80°48'W). Externally, foliage was brown, necrotic, and did not abscise; internally, sapwood was streaked with dark gray-to-bluish discoloration; and, in dead trees, holes of natal galleries of the vector from which columns of frass were attached were evident. On a semiselective medium for R. lauricola, a fungus with the pathogen's phenotype was isolated from symptomatic sapwood. Colonies were slow growing, light cream in color, with dendritic, closely appressed mycelium and often a slimy surface. A representative strain of the fungus was further identified with PCR primers for diagnostic small subunit (SSU) rDNA (1) and its SSU sequence (100% match, GenBank Accession No. JN578863). In each of two experiments, plants of 'Simmonds' avocado, the most important cultivar in Florida, were inoculated with three strains of the fungus, as described previously (3). Symptoms of laurel wilt developed in all inoculated plants and the fungus was recovered from each. After aerial and further ground surveys, additional symptomatic swampbay trees, some of which had defoliated, were detected in the vicinity of the original site. Since swampbay defoliates only a year or more after symptoms develop (4), the 2010 detection of X. glabratus may have coincided with an undetected presence of the disease. As of July 2011, a 6-km-diameter disease focus was evident in the area, the southernmost edge of which is 5 km from the nearest commercial avocado orchard. In August 2011, a dooryard avocado tree immediately north of the above focus was affected by laurel wilt, and an SSU sequence confirmed the involvement of R. lauricola (GenBank Accession No. JN613280). The outbreak of laurel wilt in Miami-Dade County represents a 150 km southerly jump in the distribution of this disease in the United States ( http://www.fs.fed.us/r8/foresthealth/laurelwilt/dist_map.shtml ) and is the first time this disease has been found in close proximity to Florida's primary commercial avocado production area. Approximately 98% of the state's commercial avocados, worth nearly $54 million per year, are produced in Miami-Dade County. Since effective fungicidal and insecticidal measures have not been developed for large, fruit-bearing trees, mitigation efforts will focus on the rapid identification and destruction of infected trees (3,4). References: (1) T. J. Dreaden et al. Phytopathology 98:S48, 2008. (2) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (3) R. C. Ploetz et al. Plant Dis. 95:977, 2011. (4) R. C. Ploetz et al. Recovery Plan for Laurel Wilt of Avocado. National Plant Disease Recovery System, USDA, ARS, 2011.

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.

First Report of Uredo manilensis in the Western Hemisphere.

Crepe jasmine, Tabernaemontana divaricata (L.) R. Br. ex Roem. & Schult. (Apocynaceae), is a popular flowering shrub in South Florida. A native of Southeast Asia, it is one of approximately 100 ornamental species in the genus. In December 2007, rust was observed on the leaves of landscape plants in Key West and Miami. The rust has become prevalent and severely affects young and old leaves of plants in the landscape and in commercial nurseries. Leaf lesions begin as chlorotic flecks that expand into necrotic spots with orange-to-reddish brown, subepidermal uredinia; brown telia develop on the abaxial side of leaves. Urediniospores are one-celled, initially hyaline, minutely echinulate and spherical, turn dark orange, and measure (22) 24 to 29 (32) × (19) 21 to 24 (26) µm. Teliospores are (26) 29 to 36 (38) × (20) 22 to 26 (28) µm, two-celled, ellipsoidal to ovoid, echinulate, constricted at the septum, reddish brown, and have 0.8-µm thick spore walls; pedicels are 25 × 5.6 µm, persistent, and hyaline. Attributes for urediniospores are consistent with those from the original description of Uredo manilensis Syd. & P. Syd. on T. coronariae in Manila (2); however, there are no reports of a telial stage for this rust. Attributes for urediniospores of the South Florida fungus were also consistent with those on herbarium specimens of U. manilensis from the U.S. National Fungus Collection, also collected in Manila but from T. polygama (BPI Accession Nos. 0155269 and 0155270). Notably, these specimens contained telia that matched those found in South Florida. Subsequent comparisons were made with herbarium specimens of the three Puccinia spp. that have been reported on Tabernaemontana spp. (the U.S. National Fungus Collection or the Arthur Herbarium, Purdue University, West Lafayette, IN). Puccinia engleriana (five specimens from India, New Guinea, and the Philippines) differs from the BPI specimens of U. manilensis and the South Florida fungus by its bigger teliospores (32) 35 to 41 (45) × (21) 22 to 24 µm. P. tabernaemontana (six specimens from Uganda) has bigger urediniospores ([45] 34 to 41 × [34] 26 to 32 µm) and yellow-brown, poorly echinulated to almost smooth teliospores. The revised material of P. morobensis (type) was poor, but according to the original description (1) and notes found in the herbarium specimen, the teliospores (24 to 29 × 33 to 45 µm) and urediniospores are larger (23 to 28 × 29 to 35 µm) and the teliospores walls are finely and sparsely echinulated to sometimes smooth, and the pedicels are very short and fragile. A specimen of the South Florida fungus was deposited with the U.S. National Fungus Collections (BPI Accession No. TBA). To our knowledge, this is the first report of U. manilensis in the Western Hemisphere and the first time a telial stage (provisionally P. manilensis) has been recognized for the fungus. This disease has become a concern in South Florida for gardeners as well as producers who must now treat the crop with fungicides. References: (1) G. B. Cummins. Mycologia. 33:148, 1941. (2) H. Sydow, and P. Sydow. Ann. Mycol. 8:36, 1910.

First Report of Tar Spot on Orange Geiger, Cordia sebestena, Caused by Diatractium cordianum in Florida.

In July 2007, tar spot symptoms were observed on the leaves of orange Geiger, Cordia sebestena L. (Boraginaceae), in the landscape and a commercial nursery in Homestead, FL. The disease appears to be spreading and is locally severe. Symptoms were circular, slightly hypertrophied spots approximately 5 to 8 cm in diameter, which were slightly chlorotic on the abaxial surface and had numerous circular blackened stroma, 0.2 to 0.4 mm in diameter, on the adaxial surface. As leaves aged and yellowed, the areas around the spots remained pale green. Embedded in the stroma were numerous perithecia, 173 to 312 µm in diameter, circular to irregular in shape, with lateral necks as much as 200 µm long and 73 to 104 µm in diameter. Asci, 77 to 92 × 11 to 13 µm, contained elongate, two-celled ascospores, 50 to 61 × 3 to 5 µm that had a conspicuous constriction at the dividing cell wall. These dimensions and the pathogen's appearance matched closely with those published for Diatractium cordianum (Ellis & Kelsey) Syd (1). Young, symptomless leaves of C. sebestena were sprayed to runoff with a suspension of ascospores approximately 104 ml-1 that were harvested from affected leaves. Inoculated leaves were placed on water-saturated paper towels in petri plates and maintained in a growth chamber at 25°C with fluorescent light at 10 h day-1. Symptoms similar to those observed on affected trees in the landscape began to develop after 21 days and perithecia were evident after 28 days. An ITS 1, ITS 2, and 5.8s rDNA sequence was deposited in GenBank (Accession No. EU541488). A herbarium specimen was deposited at the U.S. National Fungus Collections (BPI No. 878441). This is a new host record for D. cordianum and is the first time the pathogen has been reported in the United States. Previous records were from Venezuela and several Caribbean islands, including Cuba and Jamaica. Symptoms of this disease have not been observed on Texas wild olive, Cordia boissieri, in close proximity to affected C. sebestena. P. F. Cannon (1) indicated that the disease had no economic impact. However, the conspicuous nature of symptoms on C. sebestena and the importance of this tree in the South Florida ornamental trade (2) suggest that this disease may become significant on the latter host. References: (1) P. F. Cannon. Mycol. Res. 92:327, 1989. (2) E. F. Gilman and D. G. Watson. Fact Sheet ST-182. Univ. Fla, Fla Coop Ext. Serv., 1993.

First Occurrence of Anthracnose Caused by Colletotrichum gloeosporioides on Pitahaya.

Pitahaya, Hylocereus undatus Britt. & Rose, is a columnar, climbing cactus that produces a commercially important fruit. In December 2004, a new disease was found on the crop in Miami-Dade County, FL. Reddish brown lesions with conspicuous chlorotic haloes developed concentrically on the edges of vine ribs. Lesion centers became white and coalesced to rot much of the vine column, and in severe cases, only the vascular column in the vine center was not diseased. Salmon-colored spores and waxy, subepidermal acervuli, typically with setae and simple, short, erect conidiophores, were observed in lesion centers. Tissue from lesion margins was surface disinfested and plated on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI). Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. was isolated from all samples. Colonies produced abundant conidia that were hyaline, one celled, straight, cylindrical, and averaged 14.7 × 5.0 µm with ranges of 12.5 to 17.5 × 3.8 to 7.5 µm (1). Cultural and morphological characteristics of isolates matched those for C. gloeosporioides except for appressoria and hyphopodia (1,2); pitahaya isolates had a spherical rather than lobed hyphopodia reported for C. gloeosporioides and averaged 10.9 (8.5 to 12.7) × 9.1 (7.1 to 10.3) µm. Internal transcribed spacer sequences for the pitahaya isolates were nearly identical (98% homology) to those for C. gloeosporioides isolates occurring on Euphatorium thymifolia in Thailand (GenBank Accession No. AY266393). Koch's postulates were examined in greenhouse trials at the Tropical Research and Education Center, Homestead, FL. Treatments consisted of a noninoculated control, four C. gloeosporioides isolates, and an Alternaria sp. All isolates came from symptomatic pitahaya tissue collected in Miami-Dade County. Fungi were grown on PDA for 7 days at 27°C. A sterile dissecting needle was used to gently pinprick the epidermis of the stem and 2-mm-diameter plugs of C. gloeosporioides, an Alternaria sp., or clean PDA were placed over wounds. Plants were placed in a plastic tent in a greenhouse where the temperature was held at 25°C, and free moisture was maintained on plant surfaces with a household humidifier for 48 h following inoculation. Two isolates of C. gloeosporioides were shown, in repeated greenhouse experiments, to cause reddish brown lesions with conspicuous chlorotic haloes that coalesced to rot much of the vine column, and Koch's postulates were completed with the reisolation of isolates that were used to inoculate plants. The age of vine segments had no significant effect on lesion development. To our knowledge, this is the first report of C. gloeosporioides as a pathogen of pitahaya. References: (1) J. A. Bailey and M. J. Jeger. Colletotrichum: Biology, Pathology and Control. CAB International, Wallingford, UK, 1992. (2) M. Du et al. Mycologia 97:641, 2005.

First Report of Fusarium Wilt Caused by Fusarium oxysporum on Roselle in the United States.

Roselle, Hibiscus sabdariffa var. sabdariffa, is an annual that is grown primarily for its inflated calyx, which is used for drinks and jellies. It is native from India to Malaysia, but was taken at an early date to Africa and is now widely grown in the tropics and subtropics (2). In late 2005, dying plants were noted by a producer in South Florida. Plants wilted, became chlorotic, and developed generally unthrifty, sparse canopies. Internally, conspicuous vascular discoloration was evident in these plants from the roots into the canopy. After 5 days on one-half-strength potato dextrose agar (PDA), salmon-colored fungal colonies grew almost exclusively from surface-disinfested 5 mm2 pieces of vascular tissue. On banana leaf agar, single-spored strains produced the following microscopic characters of Fusarium oxysporum: copious microconidia on monophialides, infrequent falcate macroconidia, and terminal and intercalary chlamydospores. Partial, elongation factor 1-α (EF1-α) sequences were generated for two of the strains, O-2424 and O-2425, and compared with previously reported sequences for the gene (3). Maximum parsimony analysis of sequences showed that both strains fell in a large, previously described clade of the F. oxysporum complex (FOC) that contained strains from agricultural hosts, as well as human clinical specimens (2; clade 3 in Fig. 4); many of the strains in this clade have identical EF1-α sequences. Strains of F. oxysporum recovered from wilted roselle in Egypt, O-647 and O-648 in the Fusarium Research Center collection, were distantly related to the Florida strains. We are not aware of other strains of F. oxysporum from roselle in other international culture collections. Roselle seedlings were inoculated with O-2424 and O-2425 by placing a mycelial plug (5 mm2, PDA) over a small incision 5 cm above the soil line and then covering the site with Parafilm. Parafilm was removed after 1 week, and plants were incubated under ambient temperatures (20 to 32°C) in full sun for an additional 5 weeks (experiment 1) or 7 weeks (experiment 2). Compared with mock-inoculated (wound + Parafilm) control plants, both O-2424 and O-2425 caused significant (P < 0.05) vascular disease (linear extension of discolored xylem above and below wound site) and wilting (subjective 1 to 5 scale); both isolates were recovered from affected plants. F. oxysporum-induced wilt of roselle has been reported in Nigeria (1) and Malaysia (4) where the subspecific epithet f. sp. rosellae was used for the pathogen. We are not aware of reports of this disease elsewhere. To our knowledge, this is the first report of F. oxysporum-induced wilt of roselle in the United States. Research to determine whether the closely related strains in clade 3 of the FOC are generalist plant pathogens (i.e., not formae speciales) is warranted. References: (1) N. A. Amusa et al. Plant Pathol. J. 4:122, 2005. (2) J. Morton. Pages 81-286 in: Fruits of Warm Climates. Creative Resource Systems, Inc., Winterville, NC, 1987. (3) K. O'Donnell et al. J. Clin. Microbiol. 42:5109, 2004. (4) K. H. Ooi and B. Salleh. Biotropia 12:31, 1999.

Mango malformation disease and the associated fusarium species.

ABSTRACT Mango malformation disease (MMD) occurs in Asia, Africa, and the Americas and was first reported in India in 1891. The vegetative form of MMD was first reproduced in 1966 with Fusarium moniliforme and the floral form with isolates of F. moniliforme var. subglutinans from both vegetative shoots and floral tissue. The fungi were subsequently recognized as F. subglutinans. In 2002, a new species, F. mangiferae, was established based on nuclear and mitochondrial DNA sequences; it included strains of F. subglutinans from Egypt, Florida, Israel, Malaysia, and South Africa, some of which had been shown to cause MMD by artificial inoculation. At least three additional taxa have been associated with MMD: F. sterilihyphosum from Brazil and South Africa, and Fusarium sp. nov. and F. proliferatum (teleomorph: Gibberella intermedia) from Malaysia. To date, Koch's postulates have not been completed with them. In the future, gene sequencing will be essential to identify the Fusarium spp. that are associated with MMD. Work remains to be done on the morphology, sexual compatibility, pathogenicity, and toxigenicity of these taxa.

Mycosphaerella fijiensis, Causal Agent of Black Sigatoka of Musa spp. Found in Puerto Rico and Identified by Polymerase Chain Reaction.

Black Sigatoka, also known as black leaf streak, is caused by Mycosphaerella fijiensis Morelet (anamorph Pseudocercospora fijiensis (Morelet) Deighton). It is the most significant disease of bananas and plantains (Musa spp.) because most of the economically important cultivars of exported and staple commodities are highly susceptible. The Caribbean is one of the few regions of the world where black Sigatoka is not widespread. Black Sigatoka has been reported in the Bahamas, Cuba, Hispaniola, and Jamaica (2). Yellow Sigatoka, caused by M. musicola Leach (anamorph P. musae (Zimm.) Deighton), has been recognized in Puerto Rico since 1938-1939 (3). In August 2004, symptoms resembling black Sigatoka were first observed in Añasco, Puerto Rico by extension personnel from the University of Puerto Rico. Since black and yellow Sigatoka produce similar disease symptoms, a survey was conducted in the western banana- and plantain-production region of Puerto Rico to confirm the presence of black Sigatoka. Leaf samples were collected from production fields near the towns of Las Marias, Maricao, and Añasco. Single-ascospore isolates were recovered using the discharge technique from moistened pseudothecia in necrotic lesions that were inverted over water agar, and ascospores were transferred to potato dextrose agar. The isolates were subcultured in potato dextrose broth for mycelium production. DNA was isolated from mycelium with the FastDNA kit (Q-Biogen, Irvine, CA) for 19 isolates. Internal transcribed spacer as well as the 5.8s rDNA regions were polymerase chain reaction amplified with primers specific to M. fijiensis or M. musicola (1). Amplification products (˜1,100 bp) were observed for 18 of the 19 isolates, 6 of which were M. fijiensis and the remaining 12 were M. musicola, while the positive controls for both species were also amplified with the respective primer pairs. M. fijiensis was recovered from production fields close to all three towns. The source of M. fijiensis in Puerto Rico is unclear, but it may have originated from introduced leaf material and/or wind dispersed ascospores from neighboring countries. The presence of black Sigatoka in Puerto Rico will most likely increase production costs where fungicide applications will be needed to maintain yields. The USDA-ARS, Tropical Agriculture Research Station is the official Musa spp. germplasm repository for the National Plant Germplasm System. As such, efforts are underway to introduce and evaluate black Sigatoka disease-resistant clones that can satisfy local and export market criteria. References: (1) A. Johnasen. Detection of Sigatoka leaf spot pathogens of banana by the polymerase chain reaction. Chatman, UK, Natural Resource Institute, 1997. (2) R. C. Ploetz. Plant Dis. 88:772, 2004. (3) R. H. Stover. Trop. Agric. Trinidad. 39:327, 1962.