First Report of Black Spots on Avocado Fruit Caused by Neofusicoccum parvum in Mexico.

Avocado (Persea americana L.) production for export markets has increased in Mexico during the past 10 years. The production system, however, is affected by several sanitation factors, including diseases. During the spring of 2009, smooth, black, circular spots were noted on the surface of avocado fruit. A study was conducted during the winter of 2010 to ascertain the etiology and identify the fungus associated with black spot symptoms on avocado fruit in orchards of Nuevo Parangaricutiro County (19°25'00″ and 102°07'43″) in Michoacan, Mexico. Several fungal isolates were obtained on potato dextrose agar (PDA) from the margin of lesions on immature fruit. The internal transcribed spacer region (ITS) of the rDNA from representative isolates was sequenced with universal primers ITS5 and ITS4 (2). BLAST searches in GenBank showed 100% similarity of the nucleotide sequences with Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, GenBank Accession Nos. GU188001 to GU188007 and GU187985 to GU187987. A representative nucleotide sequence of this region was deposited in GenBank under the Accession No. JN203129. Strains of N. parvum produced aerial and compact mycelium on acidified PDA, the anamorph state of Botryosphaeria parva. Mycelium was initially white, turning gradually gray to black. Conidia were one celled, hyaline, ellipsoidal to fusiform, externally smooth, thin walled, nonseptate, with one or two septa with age, and an average length and width of 14.5 (9.5 to 19) × 5.8 (4.0 to 7.2) µm (n = 100). Pathogenicity tests were conducted with six avocado fruit cv. Hass. Fruit were inoculated at three evenly spaced locations on the fruit surface, either by wounding the tissue with a needle that had been dipped in a conidial mass from an 8-day-old monoconidial culture of N. parvum strain CIAD-021-11 or by placing 5 µl of 1 × 106 conidia ml-1 suspension on each inoculation site. Inoculated fruit were maintained in a moist chamber at 25°C for 2 weeks. Black lesions appeared on all wounded sites 2 days postinoculation (dpi) and on unwounded sites 4 dpi. The delay of symptom development was likely due to penetration through the lenticels, which took longer to initiate infection. No symptoms were observed in the control fruit. The pathogen was reisolated from the lesions of all inoculated fruit, thus fulfilling Koch's postulates. The results confirmed the pathogenic potential of this fungus and indicated its possible involvement in the etiology of black spot on avocado fruit. N. parvum is a cosmopolitan, plurivorous pathogen causing disease in several hosts of economic importance, such as grapes and kiwi, as well as causing stem-end rot of avocado fruit in New Zealand (1) and avocado twigs in Spain (3). To our knowledge, this is the first report of N. parvum causing black spots on avocado fruit in Mexico. References: (1) W. F. T. Hartill et al. N.Z.J. Crop Hortic. Sci. 30:249. 2002. (2) T. J. White et al. Page: 315 in: PCR Protocols: A Guide to Methods and Application. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990. (3) T. Zea-Bonilla et al. Plant Dis. 91:1052, 2007.

First Report of Leaf Rust of Blueberry Caused by Thekopsora minima in Mexico.

Blueberry (Vaccinium corymbosum L.) is becoming an important crop in the states of Jalisco and Michoacan in Mexico. Leaf rust, a disease causing extensive defoliation on plants with severe infections, was observed in the autumn of 2007 and it has become one of the most significant diseases of blueberry in these states. Symptoms on the upper surfaces of leaves appear as small, yellow spots that later turn necrotic as they enlarge and coalesce and eventually cover large areas of individual leaves. On the undersides of leaves, small flecks surrounded by small water-soaked halos appear, turn yellow, and produce powdery sori that are uredinia with urediniospores. Uredinia were hypophyllous, scattered to gregarious and at times superficially appearing confluent, up to about 300 µm in diameter, dome shaped and peridium hemispherical in cross section, orangish, becoming pulverulent, lacking obviously enlarged, well-differentiated ostiolar cells. Urediniospores were subglobose, obovate, oblong or ellipsoid, 17.6 to 27.2 × 12.8 to 17.6 µm, with hyaline, echinulate walls that are 1.2 to 1.8 µm thick, and with yellow-to-hyaline contents. Telia were not observed. On the basis of uredinial morphology (3,4), the rust was identified as Thekopsora minima P. Syd. & Syd. To distinguish this rust from other rust species causing disease on Vaccinium (2,3), a 1,414-bp region consisting of ITS2 and the 5' end of the 28S was amplified with primers Rust2inv/LR6 from uredinial lesions on infected leaves of V. corymbosum 'Biloxi' and sequenced (BPI 880580; GenBank Accession No. HM439777) (1). Results of a BLAST search of GenBank found 100% (1,414 of 1,414) identity to T. minima (GenBank Accession No. GU355675) from South Africa (3). Pathogenicity tests were completed as follows: (i) during the autumn of 2009, rusted leaves of cvs. Biloxi and Sharpblue were collected from the field; (ii) mature leaves from healthy plants of both blueberry cultivars were surface disinfested with 1% sodium hypochlorite for 2 min and rinsed with sterile distilled water; (iii) fresh urediniospores from rusted leaves were brushed directly onto the undersides of disinfested detached leaves; (iv) to avoid drying, wet cotton balls were placed on the petioles of inoculated leaves that were subsequently placed in resealable plastic bags; and (v) leaves were then incubated in a growth chamber at 22°C with a 12-h photoperiod. For each cultivar, 20 leaves were inoculated and five uninoculated leaves were included as controls and the test was repeated once. Yellow uredinia were observed 13 and 10 days after inoculation in cvs. Biloxi and Sharpblue, respectively. Leaf symptoms and uredinial characters were the same as observed previously in the field. To our knowledge, this is the first report of T. minima in Mexico. This report is significant for growers who need a diagnosis to control the disease and for breeders and plant pathologists who should consider developing more resistant cultivars. References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) F. L. Caruso and D. C. Ramsdell, eds. Compendium of Blueberry and Cranberry Diseases. The American Phytopathological Society, St. Paul, MN, 1995. (3) L. Mostert et al. Plant Dis. 94:478, 2010. (4) P. Sydow and H. Sydow. Monographia Uredinearum. Vol. III. Fratres Borntraeger, Leipzig, Germany, 1915.

First Report of Peronospora sparsa Causing Downy Mildew (Dryberry) of Rubus fruticosus in Mexico.

Michoacan State is the largest producer of blackberries (Rubus fruticosus) in Mexico with more than 4,000 ha in production. During the rainy season of 2007 (July to September), purple, angular, vein-delimited leaf spots along the midrib and major veins were observed. Affected young fruit lost their shine, became shriveled, and later dried. Some fruit split. Symptomatic leaflets from cv. Tupy were collected from the field in Tangancicuaro and Los Reyes counties. In the laboratory, 20 detached leaflets were washed with 10% bleach for 2 min, rinsed with sterile distilled water, and placed into sterile petri dishes containing 0.5% water agar. To promote sporulation of fungi, leaflets were incubated at 17°C with a 12-h photoperiod in a growth chamber. Sporangia and sporangiophores, which developed 20 days later on the underside of the leaves, were transferred to the underside of detached healthy leaves of the same cultivar with a sterile needle, and incubated as previously described. A set of noninoculated leaves were included as controls. Sporangiophores and sporangia developed on the underside of angular purple lesions on leaves 15 to 22 days after inoculation. Symptoms were identical to those observed on leaves in the field. The pathogenicity test was repeated twice with the same results. Sporangia were light brown, ovoid to elliptical, and measured 14 to 22 × 11 to 18 µm. Sporangiophores were dichotomously branched with slender curved ends. Symptoms on the leaves and fruit and oomycete morphology were similar to those described for downy mildew (2). To confirm pathogen identity, a product of ~500 bp of the nuclear ITS-rRNA was amplified from total DNA from symptomatic and asymptomatic leaves and fruit by nested PCR. The primers sets PS3/PS1 and PR3/PR4 were used for the first and second reaction, respectively (1,3). PCR products were sequenced in both directions and sequences were deposited in GenBank under Accession Nos. EU601168, EU601169, EU601170, and EU601171. Consensus sequences obtained in this study were compared with the same region of Peronospora sparsa (GenBank Accession Nos. EU391654 from Denmark and AF266783 from the UK). Similarity among these sequences varied between 99 and 100%. To our knowledge, this is the first report of downy mildew (dryberry) of blackberry caused by P. sparsa in Mexico. References: (1) B. J. Aegerter et al. Plant Dis. 86:1363, 2002. (2) W. D. Gubler. Page 15 in: Compendium of Raspberry and Blackberry Diseases and Insects. M. A. Ellis et al., eds. The American Phytopathological Society, St Paul MN, 1998. (3) H. Lindqvist et al. Plant Dis. 82:1304, 1998.

First Report of Pantoea agglomerans Causing Leaf Blight and Vascular Wilt in Maize and Sorghum in Mexico.

Zea mays and Sorghum bicolor are important crops for animal and human nutrition worldwide. In the Central Highland Valley of Mexico, both crops are extremely important, and research is aimed toward increasing yield, disease resistance, and crop adaptation from 1,900- to 2,700-m elevation. In a 3-year field breeding experiment (2004 to 2006), leaf blight and vascular wilt symptoms were frequently observed in contiguous plots of maize and sorghum crops in Montecillo, Mexico and maize plots in Tecamac, Mexico. To identify and characterize the causal agent of these symptoms, isolations were conducted on leaves from areas where healthy and diseased tissues converged. Leaf sections of 1 cm2 from both crops were disinfested, placed on casamino acid-peptone-glucose (CPG) medium, and incubated at 28°C. After 48 h, only yellow colonies were observed and 12 isolates were selected for further characterization. Physiological and biochemical tests indicated that the isolates were nonfluorescent on King's B medium, and API 50 CHE (bioMérieux, Marcy l'Etoile, France) revealed that they were negative for gelatin hydrolysis, indole production, acid production from raffinose and positive for utilization of glycerol, D-glucose, mannitol, arbutine, esculine, salicine, cellobiose, maltose, melibiose, D-fucose, and D-arabitol; all characteristics of Pantoea agglomerans. Further identification of these isolates was accomplished by DNA analysis. For DNA analysis, 1.4-kbp fragments of the 16S rRNA gene were amplified with primer set 8F/1492R (3) and sequenced with U514F/800R universal primers (2). Five sequences were obtained and deposited in GenBank (Accession Nos. EF050806 to EF050810). A phylogenetic tree was constructed using the UPGMA method (mega version 3.1). Results of the phylogenetic analysis grouped the species P. ananatis, P. stewartti, and P. agglomerans into three clusters. The five unknown sequences were grouped into the P. agglomerans cluster. There was a 98 to 99% similarity of the five 16S rRNA gene sequences with P. agglomerans strain type ATCC 27155. Pathogenicity of the 12 isolates was confirmed by injecting 108 CFU mL-1 of inoculum into stems of 3-week-old maize cv. Triunfo and sorghum cold tolerant hybrid (A1×B5)×R1 seedlings in the greenhouse at 28°C and 80% relative humidity. Also, seedlings were inoculated with water, nonpathogenic isolates of P. agglomerans from maize (GM13, and HLA1), and not inoculated as negative controls. Three replications were included for each isolate and control. All test strains developed water-soaked lesions on juvenile leaves at 8 days postinoculation and were followed by chlorotic to straw-colored leaf streaks and then leaf blight symptoms at 3 weeks postinoculation. All negative control seedlings did not develop symptoms. In addition, the 12 isolates were infiltrated at 107 CFU mL-1 into tobacco leaves that displayed a hypersensitive response at 4 days, indicating the presence of the type III secretion system (1). Isolates were reisolated, and the 16S rRNA gene fragments were 100% similar to their original isolate sequences. P. agglomerans has been reported to affect other crops, including chinese taro in Brazil (2007), onion in the United States (2006) and South Africa (1981), and pearl millet in Zimbabwe (1997); however, to our knowledge, this is the first report of P. agglomerans associated with leaf blight and vascular wilt symptoms in maize and sorghum in the Central Highland Valley of Mexico. References: (1) J. Alfano and A. Collmer. Annu. Rev. Phytopathol 42:385, 2004. (2) Y. Anzai et al. Int. J. Syst. Evol. Microbiol. 50:1563, 2000. (3) M. Sasoh et al. Appl. Environ. Microbiol. 72:1825, 2006.

First Report of the Anamorph of Glomerella acutata Causing Anthracnose on Avocado Fruits in Mexico.

Mexico is a major avocado (Persea americana) producer in the world. Glomerella cingulata (anamorph Colletotrichum gloeosporioides) has been reported as a causal agent of anthracnose on avocado fruits worldwide (3), while G. acutata (anamorph Colletotrichum acutatum) has been identified as the cause of this disease only in New Zealand (2) and Australia (4). This study was done with the objective to determine the Glomerella spp. involved as the causal agents of avocado anthracnose in Mexico. From 2003 to 2006, avocado fruits cv. Hass with anthracnose symptoms appearing as brown-black lesions on the pericarp and soft rot in the mesocarp were collected in 10 counties in Michoacan, the leading avocado-producing Mexican state. Glomerella spp. were isolated on potato dextrose agar (PDA) for molecular and morphological identification. A phylogenetic analysis was done by amplifying the internal transcribed spacer region of rDNA for 28 of the isolates. Primers ITS5/NL4 was used and successfully amplified bands of approximately 1,000 bp. Each sequence corresponding to Glomerella spp. was compared with sequences deposited in the GenBank database using BLAST. The results from molecular approach indicated 86% of the isolates used in this study were G. cingulata and 14% were G. acutata. Sequences of both species were deposited in GenBank under Accession Nos. EF221828, EF221829, and EF221830 for G. cingulata and EF175780, EF221831, and EF221832 for G. acutata. Colonies of G. acutata that developed on PDA medium were pale gray, occasionally the lower surface was olive green, and the center was covered with orange-to-salmon pink masses of conidia and perithecia. Conidia grown in the same media were straight, fusiform, 8.2 to 16.5 µm long, and 2.7 to 4.0 µm wide (4). Pathogenicity tests of G. acutata were carried out by inoculating six healthy cv. Hass fruits (1) at three evenly spaced locations on the fruit surface with a needle dipped in a conidial mass from a 3-day-old monoconidial culture of G. acutata. Fruits were then incubated in a moist chamber for 3 days. Anthracnose symptoms were observed on healthy fruits inoculated with G. acutata, while control fruits inoculated with sterile water did not develop symptoms. The fungi were reisolated successfully to confirm the pathogen's identity using morphological key. To our knowledge, this is the first report of G. acutata causing anthracnose on avocado fruits in Mexico. References: (1) R. Guetsky et al. Phytopathology 95:1341, 2005. (2) W. F. T. Hartill. N. Z. J. Crop Hortic. Sci. 19:297, 1991. (3) D. Prusky. Annu. Rev. Phytopathol. 34:413, 1996. (4) J. H. Simmonds. Qld. J. Agric. Anim. Sci. 22:437, 1965.