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.

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.

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.

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.

Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies.

Panama disease of banana, caused by the fungus Fusarium oxysporum f. sp. cubense, is a serious constraint both to the commercial production of banana and cultivation for subsistence agriculture. Previous work has indicated that F. oxysporum f. sp. cubense consists of several clonal lineages that may be genetically distant. In this study we tested whether lineages of the Panama disease pathogen have a monophyletic origin by comparing DNA sequences of nuclear and mitochondrial genes. DNA sequences were obtained for translation elongation factor 1alpha and the mitochondrial small subunit ribosomal RNA genes for F. oxysporum strains from banana, pathogenic strains from other hosts and putatively nonpathogenic isolates of F. oxysporum. Cladograms for the two genes were highly concordant and a partition-homogeneity test indicated the two datasets could be combined. The tree inferred from the combined dataset resolved five lineages corresponding to "F. oxysporum f. sp. cubense" with a large dichotomy between two taxa represented by strains most commonly isolated from bananas with Panama disease. The results also demonstrate that the latter two taxa have significantly different chromosome numbers. F. oxysporum isolates collected as nonpathogenic or pathogenic to other hosts that have very similar or identical elongation factor 1alpha and mitochondrial small subunit genotypes as banana pathogens were shown to cause little or no disease on banana. Taken together, these results indicate Panama disease of banana is caused by fungi with independent evolutionary origins.

First Report of Black Sigatoka in Bolivia.

Black Sigatoka, caused by Mycosphaerella fijiensis, is the most important disease of banana worldwide (1). It affects cultivars of the Cavendish subgroup that are used for export and important, locally consumed cooking and dessert bananas and plantains, reducing yields by 50% or more. Black Sigatoka first appeared in the Western Hemisphere in 1972 in Honduras, and has spread to all other countries in Central America (1980), Mexico (1980), and the following islands in the Caribbean: Cuba (1992), Hispanola (Dominican Republic) (1996), and Jamaica (1994). In South America, the disease has spread to Colombia (1981), Ecuador (1986), Venezuela (1992), and Peru (1994) (1). In June 1996, symptoms of the disease were observed in the San Carlos area in the western Chapare region of Bolivia. During surveys conducted in March and June 1997, several Cavendish clones, Dulce Cajita (Pisang mas), Guineo (Silk), Morado (Red), and Platano (French and Horn plantain) were affected. In each of eight major banana-producing areas in the region, disease incidence and severity were recorded at several representative sites on Cavendish cultivars, which were the most widely spread and susceptible clones in the region. Disease incidence was 100% in all areas from San Carlos to Ingavi B, 30 km to the east, and disease severity, rated as the youngest leaf spotted (YLS), ranged from means of 4.5 to 8 in the same areas. The disease was less common or rare in the Valle Sajta area, 60 km east of San Carlos, the reserve of the Yuqui indigenous group at the confluence of the Rio Chimore and Rio Useuta, 15 km northeast of San Carlos, and the southernmost settlements of the Yuracare indigenous group on the Rio Chapare, 20 km north of Ingavi B (incidences = 0 to 50%). Symptoms began as brown streaks on the abaxial leaf surface, 1 to 3 mm in length, and became visible on the adaxial surface and enlarged to wet, dark brown streaks, 1 to 2 × 10 to 20 mm, with chlorotic haloes. Ultimately, large portions of the leaf became blackened and watersoaked. The presence of the disease in the San Carlos, Ingavi B, and Senda B areas and the Yuqui reserve was confirmed after microscopic examination of the anamorph, Paracercospora fijiensis, on affected leaf tissue: scars were present on the base of conidia, and only simple conidiophores were found (2). This is the first report of black Sigatoka in Bolivia, and represents the southernmost extent of the disease on the South American continent. High rainfall in western portions of the Chapare (4 to 7 meters per year) makes it unlikely that the disease could be controlled effectively or economically in the region with fungicides. We believe this is the closest approach of the disease to Brazil (ca. 700 km), the last major banana-producing country in which black Sigatoka has not been reported. Moreover, the outbreaks in the northern Chapare are thought to be the first across the colonist frontier to indigenous Amazonian populations that rely on plantains and bananas as staple foods. References: (1) X. Mourichon and R. A. Fullerton. Fruits 45:213, 1990; (2) N. Pons. Trans. Br. Mycol. Soc. 89:120, 1987.