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 King's 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.

Root Rot of Hydroponically Grown Lettuce Caused by Phytophthora drechsleri in Mexico.

During March of 2008, bibb lettuce (Lactuca sativa L.) plants with severe wilting and root rot were observed in a commercial liquid-hydroponic greenhouse in Guanajuato, Mexico. By July of that year, the disease affected most plants in the facility. A Phytophthora sp. was consistently isolated from diseased roots on potato carrot agar. Several Phytophthora isolates were morphologically characterized. Sporulation was achieved by placing colonized disks of clarified V8 juice agar (V8A) into nonautoclaved soil extract (10 g avocado soil/1,000 ml distilled water, stirred for 3 h, and filtered). Sporangia were persistent, nonpapillate, and 40 to 58 µm long × 30 to 40 µm wide. External and internal proliferation was observed. Hyphal swellings were predominantly rounded. Oospores were not observed. The isolates grew on V8A at 35°C. Pathogenicity tests were conducted twice by utilizing a representative isolate (AC1) on bibb lettuce seedlings (10 replicates per experiment). Seeds were placed on sterile, water-soaked paper in petri dishes. After 10 days, each lettuce seedling was placed into a tube containing approximately 2 ml of sterile distilled water and 2,000 zoospores. Control plants were placed in tubes with water only. Plants were incubated for 7 days in a moist chamber at 25°C. Symptoms of wilting and root necrosis were observed 2 to 3 days after inoculation. All plants were dead 5 to 7 days after inoculation. A Phytophthora sp. was always isolated from the roots of inoculated plants. Control plants remained healthy. The pathogen was identified as Phytophthora drechsleri Tucker according to morphological characteristics. To confirm the identity of the pathogen, sequences of the internal transcribed spacers (ITS) were obtained from three representative isolates. The ITS sequences that were obtained shared 100% homology to several strains of P. dreschleri, including isolates from cucurbits (GenBank Accession No. AF228097). The ITS sequence was deposited in NCBI as Accession No. FJ790770. P. cryptogea and P. dreschleri have been reported as causing root rot on lettuce grown hydroponically in the United States and Korea (1,2). To our knowledge, this is the first report of P. drechsleri causing root rot on lettuce in Mexico. References: (1) H. J. Jee et al. Plant Pathol. J. 17:311, 2001. (2) A. R. Linde et al. Plant Dis. 74:1037, 1990.

First Report of Powdery Mildew on Greenhouse Tomatoes Caused by Oidium neolycopersici in Michoacan, Mexico.

During June and July of 2007, powdery mildew-infected tomato (Lycopersicum esculentum Mill. cv. Reserve) plants were observed in a commercial greenhouse with an open hydroponic system in Morelia County. Disease incidence increased from 0.5% to more than 90% in 1 month. Infected plants showed leaves with irregular areas of dense, white mycelium covering most of the upper surface. Microscopic analysis showed hyaline, septate hyphae with lobed appressoria. Conidia were ellipsoid to ovoid and 30 to 45 (38) µm × 15 to 20 (16) µm. Conidiophores were erect, 80 to 120 (103) µm, consisted of a foot cell 42 to 67 (56) µm, and two to three short cells. Conidia were produced singly. On the basis of the observed morphological characteristics, the fungus was identified as Oidium neolycopersici L. Kiss (1). Pathogenicity tests were conducted on fourth true-leaf tomato seedlings cv. Reserve under greenhouse conditions (22 ± 5°C). Inoculation was performed by transferring conidia from infected leaves to the leaves of uninfected tomato seedlings with a single-edged razor blade. Powdery mildew symptoms began to develop 7 days after inoculation. Symptoms and morphological characteristics were similar to those observed in the commercial greenhouse. Noninoculated plants remained healthy throughout the experiments. To our knowledge, this is the first report of O. neolycopersici causing powdery mildew on tomato in Michoacan, Mexico. This disease has been reported from Canada, Europe, Japan, the United States (2), and Venezuela (3) on greenhouse and field tomato crops. The observed high incidence and severe infection indicates that this disease may become an important problem in greenhouse tomatoes in Mexico. References: (1) L. Kiss et al. Mycol. Res. 105:684, 2001. (2) L. Kiss et al. Plant Dis. 89:491, 2005. (3) J. O. Montilla et al. Plant Dis. 91:910, 2007.

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.

Population Structure of Phytophthora infestans in the Toluca Valley Region of Central Mexico.

ABSTRACT We tested the hypothesis that the population of Phytophthora infestans in the Toluca valley region is genetically differentiated according to habitat. Isolates were sampled in three habitats from (i) wild Solanum spp. (WILD), (ii) land-race varieties in low-input production systems (RURAL), and (iii) modern cultivars in high-input agriculture (VALLEY). Isolates were sampled in 1988-89 (n= 179) and in 1997-98 (n= 389). In both sampling periods, the greatest genetic diversity was observed in RURAL and VALLEY habitats. Based on the Glucose-6-phosphate isomerase and Peptidase allozymes, the subpopulations from the three habitats were significantly differentiated in both sampling periods. In contrast to allozyme data for 1997-98, no differences were found among the three subpopulations for sensitivity to metalaxyl. Two groups of isolates identical for allozyme and mating type were further investigated by restriction fragment length polymorphism fingerprinting; 65% of one group and 85% of another group were demonstrated to be unique. The genetic diversity data and the chronology of disease occurrence during the season are consistent with the hypothesis that populations of P. infestans on wild Solanum populations are derived from populations on cultivated potatoes in the central highlands of Mexico near Toluca.

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.