Sylvia Patricia Fernández-Pavía

Identification and Characterization of a Novel Etiological Agent of Mango Malformation Disease in Mexico, Fusarium mexicanum sp. nov.

The primary objective of this study was to characterize Fusarium spp. associated with the economically devastating mango malformation disease (MMD) in Mexico. In all, 142 Fusarium strains were isolated from symptomatic mango inflorescences and vegetative tissues in eight geographically diverse Mexican states from 2002 through 2007. Initially, all the Mexican isolates were screened for genetic diversity using appolymerase chain reaction and random amplified polymorphic DNA markers and were grouped into seven distinct genotypes. Based on results of these analyses, evolutionary relationships and species limits of the genetically diverse MMD-associated Fusarium spp. were investigated using multilocus DNA sequence data and phylogenetic species recognition. Maximum parsimony analyses of a five-locus data set comprising 5.8 kb of aligned DNA sequence data indicated that at least nine phylogenetically distinct Fusarium spp. within the Gibberella fujikuroi species complex are associated with MMD, including one species within the African clade (Fusarium pseudocircinatum), two species within the Asian clade (F. mangiferae and F. proliferatum), and at least six species within the American clade (F. sterilihyphosum and five undescribed Fusarium spp.). Molecular phylogenetic analyses indicate that a novel genealogically exclusive lineage within the American clade was the predominant MMD associate in Mexico. This new Fusarium sp. caused MMD and could be distinguished from all other known species morphologically by the production of mostly sterile, coiled hyphae which are typically associated with sporodochial conidiophores together with unbranched or sparsely branched aerial conidiophores. Kochs postulates were completed for isolates of the new species on nucellar seedlings of mango cv. Ataulfo. This pathogen is formally described herein as F. mexicanum.

Soilborne Oospores of Phytophthora infestans in Central Mexico Survive Winter Fallow and Infect Potato Plants in the Field

Survival and infectivity of oospores in soils naturally infested with P. infestans oospores were studied in central Mexico. Sporangia were selectively eliminated from soil samples to determine infectivity attributable to the presence of oospores. Selective elimination of sporangia was achieved by two cycles of wetting and drying the soil. Oospore concentration, viability, and infectivity varied among soils collected during the winter fallow in different locations of central Mexico. In some soils, oospores were infective regardless of the time at which they were collected during the winter fallow. However, oospore viability and infectivity decreased following 2 years of intercropping. The number of stem lesions and initial disease severity were significantly higher in soils with moderate (20 to 39 oospores g-1 soil) oospore infestation compared with soils with low (0 to 19 oospores g-1 soil) infestation. Our study confirms that oospores can survive winter fallow and serve as a source of primary inoculum in the central highlands of Mexico. Oospore survival appeared lower in the Toluca Valley soil, which may be an indication of soil suppressiveness.

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, β-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 Kochs 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.

Formation of Phytophthora infestans Oospores in Nature on Tubers in Central Mexico

Oospore formation by Phytophthora infestans in nature has been detected on potato leaflets in central Mexico (1), but there are no reports of oospore formation on tubers. A severe late blight epidemic occurred in Calimaya, Mexico, in fields where potato cv. Alpha was planted during the summer of 2000. Yield was reduced despite numerous applications of fungicide. Four hundred potato tubers left in the field were collected from the upper 10 cm of soil and examined for late blight symptoms. Tubers with soft and dry rot symptoms were observed, but symptoms of pink rot (Phytophthora erythroseptica) were not found. Four percent of the tubers showed late blight symptoms. Sections of 10 tubers with late blight symptoms were air-dried for 2 weeks in the laboratory and homogenized with a mortar and pestle. Glycerol was added to the homogenized tissue and observed microscopically. Aplerotic oospores (10 to 15 oospores per tuber) with amphyginous antheridia typical of P. infestans were observed. P. mirabilis morphologically similar to P. infestans is present in the area but it does not infect potato tubers. The number of oospores observed in our tuber sample was much lower than the number reported on leaflets (>1,000 oospores per leaflet) in the Toluca Valley. Low numbers of oospores have been reported on tubers artificially inoculated with P. infestans under field conditions (2). Infected tubers left in the field may act as a source of primary inoculum. To our knowledge, this is the first report of oospores of P. infestans found on tubers in Mexico under natural field conditions. References: (1) M. E. Gallegly and J. Galindo. Phytopathology 48:274, 1958. (2) A. Levin et al. Phytopathology 91:579, 2001.

First report of gladiolus rust caused by Uromyces transversalis in Michoacán, México.

During October 2005, rust lesions were observed on leaves of gladiolus (Gladiolus sp.) plants being grown for flower production in a 20-ha field in eastern Michoacán, México. Disease incidence was near 100% in the field. Five symptomatic plants were collected on 11 and 25 October 2005, from each of 10 farms for further examination. Uredinia were scattered, orange, elliptical to irregular, and arranged transversely across the leaf. The sori were covered by the epidermis initially and later were erumpent and pulverulent. Urediniospores were bright yellow gold, ovate to oblong, and measured 15 × 19 μm (average). The urediniospore wall was hyaline and minutely echinulate. Telia were scattered, dark brown, elliptical, arranged transversely across the leaf, and were covered by the epidermis. Teliospores were irregularly pyriform, ovate, irregular or angular, light to dark brown with a conical or truncate apex and measured 17 × 23 μm (average). The teliospore wall measured 1 μm (average) thick at the sides and 3 μm (average) thick at the apex. Pedicels were light yellow and measured as much as 60 × 3 μm (average). On the basis of these characters, the rust was identified as Uromyces transversalis (Thüm.) G. Winter (1). To our knowledge this is the first report of U. transversalis causing gladiolus rust in Michoacán, México. Originally reported from Africa, the disease has been reported from Argentina, Brazil, southern Europe, and Oceania (1). Gladiolus rust caused by Uromyces transversalis is a quarantine disease for Europe and the United States. There have been unpublished reports of interceptions of this rust on cut flowers of gladiolus going from México into the United States (1). References: (1) J. R. Hernández. Invasive Fungi. Gladiolus Rust. Systematic Botany and Mycology Laboratory, Online publication. ARS, USDA, 2004.

First Report of Blight on Ipomoea purpurea Caused by Phytophthora ipomoeae

Several wild species of Ipomoea grow in the central highlands of Mexico. During the summer of 1999, in Metepec, Mexico, blighted leaves and petioles of Ipomoea purpurea were collected from diseased plants and placed in a moist chamber to induce sporulation. Sporangia that formed on the lesions were transferred with a piece of agar to selective rye agar medium (2). Phytophthora ipomoeae was consistently isolated. Species identification was based on sporangial and gametangial characteristics of five cultures grown on rye agar. Sporangia were mainly ellipsoid but occasionally ovoid, semipapillated, and deciduous with a short pedicel. All isolates were homothallic with smooth-walled and aplerotic oospores. Genotypic analysis for the allozymes Peptidase (Pep) and Glucose-6-phosphate isomerase (Gpi) indicated that all five isolates belonged to one genotype with alleles 78/78 (Pep) and 108/108 (Gpi). Morphological characteristics and the allozyme genotype correspond to the new, recently described species P. ipomoeae Flier & Grünwald (1) isolated from I. orizabensis (Pelletan) Ledeb. ex Steud. (I. tyrianthina) Lindl. and I. longepedunculata (Mart. & Gal.) Hemsl. Pathogenicity tests were carried out with leaves from greenhouse-grown I. purpurea plants. Detached leaves were inoculated with a suspension of 103 sporangia per ml and kept in a moist chamber at room temperature (17 ± 3°C). Lesions were observed between 7 and 15 days after inoculation and were characteristic of those observed in the field. The pathogen was reisolated from inoculated symptomatic tissue. To our knowledge, this is the first report of blight on I. purpurea caused by P. ipomoeae. References: (1) W. Flier et al. Mycol. Res. 106:848, 2002. (2) N. J. Grünwald et al. Phytopathology 91:882, 2001.

First Report of Haplotype I-b of Phytophthora infestans in Central Mexico

Central Mexico is considered a center of genetic diversity for Phytophthora infestans on the basis of a range of genotypic and phenotypic characteristics (3). Surprisingly, while mitochondrial DNA (mtDNA) haplotypes I-a, II-a, and II-b have been reported from central Mexico, haplotype I-b has not been found in central Mexico (1). Therefore, a more extensive search for haplotypes was conducted in areas where sexual reproduction occurs. During the summer of 2003, leaflets of cvs. Rosita and Tollocan with a single lesion of late blight were collected in the area of Villarreal, located in Terrenate County in Tlaxcala, Mexico (170 km northeast of Mexico City). Fourteen P. infestans isolates were characterized for mtDNA haplotype, isozyme genotype (glucose 6- phosphate isomerase [Gpi] and peptidase [Pep]), and mating type. Isolation, mating type, and isozyme genotype were characterized following reported protocols (1,4). MtDNA haplotype was determined by amplifying and digesting the P2 and P4 regions and comparing amplicons to those of reference strains of known haplotype (1,2). Twelve isolates were mtDNA haplotype I-a and two were I-b. While the mtDNA I-b has been associated with the US-1 lineage (mating type: A1, Gpi: 86/100, Pep: 92/100), the genotypes for the Mexican isolates were A2, 86/100 Gpi, 100/100 Pep from cv. Rosita and A2, 86/100 Gpi, 92/100 Pep from cv. Tollocan. To our knowledge, this is the first report of the I-b mtDNA haplotype of P. infestans from central Mexico and it is now clear that all four haplotypes exist in Mexico. This finding therefore, stresses the importance of including a representative regional sampling of Mexican and Andean isolates in studies inferring the origin of this species. References: (1) W. G. Flier et al. Phytopathology 93:382, 2003. (2) G. W. Griffith and D. S. Shaw. Appl. Environ. Microbiol. 64:4007, 1998. (3) N. J. Grünwald and W. G. Flier. Ann. Rev. Phytopathol. 43:171, 2005. (4) N. J. Grünwald et al. Phytopathology 91:882, 2001.

Late Blight Resistance of Five Mexican Potato Cultivars in the Eastern Sierra of the State of México

In the sierras of Central México there are existing cultivated potato areas as well as potential regions for commercial production and seed production. Prevailing weather conditions during the summer in these regions are also favorable for potato late blight disease (Phytophthora infestans (Mont) De Bary). The objectives of this work were to evaluate five resistant cultivars versus a susceptible control (‘Alpha’) in the eastern sierra of the State of México in order to assess resistance against the disease.Environmental conditions for potato late blight were favorable all five years of the study, and at the end of each growing cycle, Alpha had 100% of late blight severity. Cultivars Montserrat, Michoacán, Norteña and Sangema (Rosita) showed acceptable disease resistance. Disease severity on the resistant varieties varied from 2% to 20%. There was a delay of two to eight weeks in the appearance of the first symptoms in relation to Alpha cultivar. These delays were four to five times greater than the ones observed at the Toluca Valley in previous experiments. Yield varied from 94 to 1,475 g per plant and was related to resistance to late blight. This work corroborates potato late blight resistance observed by INIFAP and CEEM scientists at the Toluca Valley. The expression of resistance is considered to be of great help in controlling the disease with a lower number of fungicide applications and better profit. In addition, using these varieties and therefore reducing the number of fungicide sprays also reduces direct damage to people and the environment.ResumenEn las sierras de la Región Central de México, existen áreas actualmente cultivadas con papa y zonas con potencial para la producción comercial o para semilla. Las condiciones climatológicas de la zona prevalentes en estas regiones durante el verano son tambien favorables para el Tizón Tardío de la papa (Phytophthora infestans (Mont) De Bary). Los objetivos de este trabajo fueron: Evaluar 5 variedades resistentes y un testigo susceptible (‘Alpha’), bajo condiciones de la sierra oriental del Estado de México para determinar su resistencia a la enfermedad.En los 5 años de estudios, las condiciones ambientales para el desarrollo del Tizón Tardío de la Papa fueron favorables y al final del ciclo de crecimiento, Alpha presentó un 100% de severidad del tizón tardío. Los cultivares Montserrat, Michoacán, Norteña y Sangema (Rosita) mostraron una resistencia aceptable a la enfermedad. La severidad de la enfermedad en el follaje de las variedades resistentes varió de 2% a 20%. Hubo un retraso de 2 a 8 semanas, en la aparición de los primeros síntomas en relación con el cultivar Alpha. Estos retrasos fueron de 4 a 5 veces mas grandes que los observados en experimentos previamente efectuados en el Valle de Toluca. El rendimiento varió de 94 to 1,475 g por planta y estuvo relacionado con la resistencia al tízon tardío. Este trabajo corrobora la resistencia al tizón tardío de la papa observada por los científicos del INIFAP y del CEEM en el Valle de Toluca. La expresión de la resistencia es considerada de ser de gran ayuda en el control de la enfermedad con un número menor de aplicaciones de fungicidas y mas ganancias. Además, con el uso de estas variedades, al reducir el número de aspersiones de fungicidas, también se reduce el daño directo al hombre y al medio ambiente.

High levels of diversity and population structure in the potato late blight pathogen at the Mexico centre of origin

Globally destructive crop pathogens often emerge by migrating out of their native ranges. These pathogens are often diverse at their centre of origin and may exhibit adaptive variation in the invaded range via multiple introductions from different source populations. However, source populations are generally unidentified or poorly studied compared to invasive populations. Phytophthora infestans, the causal agent of late blight, is one of the most costly pathogens of potato and tomato worldwide. Mexico is the centre of origin and diversity of P. infestans and migration events out of Mexico have enormously impacted disease dynamics in North America and Europe. The debate over the origin of the pathogen, and population studies of P. infestans in Mexico, has focused on the Toluca Valley, whereas neighbouring regions have been little studied. We examined the population structure of P. infestans across central Mexico, including samples from Michoacán, Tlaxcala and Toluca. We found high levels of diversity consistent with sexual reproduction in Michoacán and Tlaxcala and population subdivision that was strongly associated with geographic region. We determined that population structure in central Mexico has contributed to diversity in introduced populations based on relatedness of U.S. clonal lineages to Mexican isolates from different regions. Our results suggest that P. infestans exists as a metapopulation in central Mexico, and this population structure could be contributing to the repeated re‐emergence of P. infestans in the United States and elsewhere.

An initial assessment of genetic diversity for Phytophthora capsici in northern and central Mexico

Phytophthora capsici causes significant damage to vegetable production in Mexico, but very little is known about the population structure or how populations survive and spread. The objective of the present study was to evaluate the genetic diversity of P. capsici isolates recovered from 1998–2014 in central and northern Mexico. Isolates (n = 81) were genotyped for 33 polymorphic single nucleotide polymorphism (SNP) loci using a targeted sequencing approach. There were a total of 72 unique genotypes and both the A1 and A2 mating types were common in both regions. Genetic analyses suggest clonal reproduction may play a more prominent role in the north, but the large proportion of unique genotypes and the finding of both mating types throughout both regions suggests outcrossing and sexual recombination likely play an important role in the overall epidemiology. Further studies with finer scale sampling at single locations over multiple years will be valuable.