Gerardo Rodríguez-Alvarado

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.

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.

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.

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.

First Report of Phytophthora capsici Causing Wilt on Hydropronically Grown Cucumber in Mexico

During August 2005, wilted cucumber (Cucumis sativus cv. Tasty Green) plants were observed in a commercial greenhouse with a closed hydroponic system in the state of Mexico. Disease incidence was 50%. Diseased plants were detected 15 days after transplanting, when plants were overwatered. Yield was severely reduced when disease affected mature plants. Wilted plants showed basal stem lesions and root rot. Phytophthora capsici was consistently isolated from diseased tissue on corn meal agar (CMA) with tartaric acid. Oomycete identification was based on sporangial and gametangial characteristics (2). Sporangia produced on blocks of CMA at 25°C were spherical, broadly ellipsoid or obovoid with one papillae, and deciduous with a long pedicel (1). The isolates were heterothallic, and oogonia with amphigynous antheridia were observed in pairings with an A1 isolate of P. capsici, therefore, the isolates were determined to be an A2. Pathogenicity tests were conducted on 2-month-old cucumber seedlings under controlled conditions (25°C). Inoculation was performed by placing small pieces of agar with mycelium of 5- to 7-day-old cultures on the stem base and wrapping with Parafilm. Control plants were inoculated with CMA agar. No symptoms were observed on the control. Plants inoculated with the P. capsici isolated from the diseased cucumbers showed a basal stem lesion, followed by wilting and death 7 to 14 days after inoculation. The isolate was also pathogenic on tomato and eggplant that were grown at the same time in the commercial greenhouse sharing the nutrient solution. P. capsici sporangia were observed on the roots of both hosts. To our knowledge, this is the first report of P. capsici affecting cucumber in a hydroponics system in Mexico. References: (1) M. Aragaki and J. Y. Uchida. Mycologia 93:137, 2001. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society. St. Paul MN, 1996.

First Report of Xanthomonas fragariae Causing Angular Leaf Spot on Strawberry Plants in México

The state of Michoacán is the most important strawberry producer in México. During January 2007, field-grown strawberry plants cv. Aromas showing vein necrosis were observed in 3 ha in Zamora County, in fruit production fields. The average disease incidence in the field was 80%. Infected plants presented water-soaked lesions limited by veins on the lower leaf surfaces, which enlarged to form angular spots (1). Additionally, most affected plants presented severe necrosis in the main veins and reddish to necrotic lesions on the upper leaf surfaces. Gram-negative bacteria were consistently isolated from leaves with water-soaked lesions. Isolated bacteria produced mucoid, yellow colonies on YDC, grew on tween and nutrient agar (NA), but not on SX media. Strains produced non-fluorescent colonies on Kings B media, were positive starch hydrolysis, negative esculin hydrolysis; and produced acid from fructose but not from arabinose, galactose, celobiose, and trehalose. Growth was inhibited by 2% NaCl (3). Indirect ELISA analysis (NEOGEN, Lansing, MI) was conducted using antibodies specific for Xanthomonas fragariae. Conventional PCR assay using the primer pairs 241A/241B was performed (2). The ELISA test was positive. The expected 300- and 550-bp bands were observed in the PCR analysis. The bacteria was identified as X. fragariae Kennedy and King. Pathogenicity tests were conducted twice in a greenhouse (24 ± 4°C) on a total of five strawberry cv. Aromas plants. The main vein of each of three leaves per plant were punctured using sterile needles. Pathogen inoculum was obtained from 6- to 8-day-old NA cultures. Bacteria were applied onto the wounds with a sterile cotton swab dipped into the bacterial suspension (105 CFU/ml). Inoculated plants were covered with plastic bags for 48 h. Symptoms resembling those seen in the field developed on all inoculated plants after 9 days. X. fragariae was re-isolated from the necrotic lesions and identified by PCR. Control plants were similarly inoculated with water but did not develop symptoms. To our knowledge, this is the first report of X. fragariae causing angular leaf spot in strawberry in Michoacán, México. References: (1) J. L. Maas, ed. Compendium of Strawberry Diseases. The American Phytopathological Society, St. Paul, MN, 1998. (2) M. R. Pooler et al. Appl. Environ. Microbiol. 62:3121, 1996. (3) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001.

Mango nurseries as sources of Fusarium mexicanum, cause of mango malformation disease in central western Mexico

Malformation is the most important disease of mango in Mexico. It affects floral and vegetative shoots, and reduces fruit production. Although several species of Fusarium cause the disease worldwide, F. mexicanum is most important in Mexico. To better understand epidemiology of disease in Mexico, we studied mango nurseries as a source of infected planting material. In 2011, 2012 and 2014, 20 mango nurseries in the states of Colima (1 nursery), Jalisco (1 nursery), Guerrero (6 nurseries) and Michoacan (12 nurseries) were examined for floral and vegetative symptoms of the disease. Although malformed plants were not observed in Colima, Guerrero and Jalisco, malformed mango seedlings and grafted plants were observed in 10 commercial nurseries in Michoacan in 2011 and 2012. Thirty-three isolates from a total of 197 symptomatic plants were identified as F. mexicanum, based on morphological and genetic criteria. Analyses of partial sequences of the EF-1α and β-tubulin genes and mating type indicated that isolates of F. mexicanum from mango nurseries are mostly a single clonal population, identical to F. mexicanum isolates previously described from mango orchards in Michoacan. Thus, mango nurseries in Michoacan could be a significant source of inoculum when new mango orchards are established in the state. Efforts should be made to produce and utilize pathogen-free planting material during orchard establishment.