First Report of Powdery Mildew (Podosphaera pannosa) of Roses in Sinaloa, Mexico.

Rose (Rosa spp.) is the most important ornamental plant cultivated in greenhouse and open fields in Mexico but its quality has been limited by powdery mildew (PM). High incidence and disease damage is common during winter in Sinaloa, Mexico (temperature range 18 to 25°C and prolonged episodes of relative humidity ≥90%). The fungus attacks leaves and flowers and grows abundantly on the pedicels, sepals, and receptacles, especially when the flower bud is unopened (2). Field advisors in Mexico have referred to Sphaerotheca pannosa (Wallr. ex Fr.) Lév. as a causal agent of the disease. However, there has not been solid scientific evidence to support this statement. Morphometric and molecular analysis were conducted to elucidate the identity of the fungal isolates collected from 2012 through 2013 in northern Sinaloa. PM specimens included eight different rose varieties. Conidiophores and conidia were observed under a compound microscope. The mycelium had a mean diameter of 4.7 to 6.0 µm; conidiophores (Euoidium type) 2 to 5 celled, occasionally 6 celled emerged from the superficial mycelium; conidiophores were unbranched with conidia produced in chains from the apex. The average length of the conidiophores was 54.9 to 98.0 µm; the foot cell of the conidiophores was straight and was 24.9 to 53.6 µm long with a diameter from 8.2 to 9.8 µm across its medium part. Conidia originated from unswollen conidiogenous cells, with fibrosin bodies, formed in long chains, and were cylindrical to ovoid, 25.8 to 30.4 µm long and 13.9 to 17.3 µm wide. The outline of the conidial chains was crenate. Conidia exhibited a slight constriction at one end. The germ tubes emerged from a shoulder of the conidia. The outer wall of partially collapsed conidia showed longitudinal and transversal wrinkling and slight constrictions at the ends; the terminal end of the conidia was concentrically ridged. For molecular characterization, the ITS region of the specimens was amplified with primers ITS1F and ITS4. Phylogenetic analysis was performed with MEGA 6.0 (bootstrap = 1,000) using Kimura 2 parameter (K2P) substitution model. The resulting phylogeny grouped our specimens (GenBank KM001665 to 69) within a clade of Podosphaera pannosa (Wall.: Fr.) de Bary (formerly known as Sphaerotheca pannosa) sequences (e.g., AB525938; bootstrap (1,000) = 98). Phylogenetic and morphometric data are in agreement with descriptions of the anamorphic P. pannosa (1,3). Morphological studies indicate that P. macularis (previously known as S. humuli) and P. pannosa are not indistinctly different (2). Phylogenetic analysis showed relationship to P. pannosa, but not to P. macularis. Typical symptoms caused by P. pannosa were observed. Morphological studies (4) reported the anamorph of P. pannosa on Rosa spp. in central Mexico. To date, no report exists on the molecular identification of P. pannosa associated to roses in northern Sinaloa, Mexico. Future research directions should focus on finding the teleomorph of the fungus to support its identity, and to explore disease management tools such as effective fungicides and developing resistant rose cultivars. References: (1) U. Braun et al. Page 13 in: The Powdery Mildews: A Comprehensive Treatise. APS Press, St. Paul, MN, 2002. (2) R. K. Horst. Compendium of Rose Diseases. APS Press, St. Paul, MN, 1983. (3) L. Leus et al. J. Phytopathol. 154:23, 2006. (4) Yañez-Morales et al. Some new reports and new species of powdery mildew from Mexico. Schlechtendalia 19:46, 2009.

First Report of Powdery Mildew (Pseudoidium anacardii) of Mango Trees in Sinaloa, Mexico.

Powdery mildew of mango is an important disease in Mexico's northern Sinaloa state. Identification of the causal fungal agent has been hindered by the absence of information regarding its teleomorph, as well as a detailed morphometric analysis of the anamorph and molecular characterization. The first symptoms of the disease appear in mango inflorescences of early February, and it subsequently affects young fruits. The disease progresses during March and early April, causing significant fruit abortion and a scabby appearance in a high percentage of fruits that remain attached to the trees. We observed the disease on inflorescences but not in leaves during our sampling period. Powdery mildew specimens were collected during 2011 and 2012 and included Kent and Keith varieties from commercial orchards, and creole materials from backyards of private residences in the Ahome and Fuerte Counties of northern Sinaloa, Mexico. Symptomatic inflorescences were analyzed morphologically. Conidiophores and conidia were prepared by touching the whitish lesions with clear adhesive tape, which was then placed over microscope slides with a drop of distilled water and observed under a compound microscope. The anamorph structures of the pathogen were measured. The mycelium was septate and ramified on the surface of the host, forming a dense coat of branching hyphae. The mycelium had a diameter of 2.5 to 8.7 µm; conidiophores (Pseudoidium type) emerged from the superficial mycelium, were unbranched, and consisted of 1 to 3 cells with conidia forming singly from the apex. The length of the conidiophores varied from 30.0 to 77.5 µm; the foot cell of the conidiophores was straight, 10.0 to 47.5 µm long and with a diameter of 5.0 to 15.5 µm across its midpoint. Conidia without fibrosin bodies were borne singly, and were ellipsoid/ovoid, 22.5 to 46.2 µm long and 15.0 to 27.5 µm wide. Eighty percent of the germ tubes were forked (lobed); the rest were simple, emerged from the end, and were occasionally on the side of the conidia. Germ tubes ranged from 2.0 to 7.2 µm at the midpoint. The surface of the conidia appeared smooth under the scanning electron microscope, and elliptical conidia appeared constricted at their ends; this, however, was not observed in the ovoid conidia. In both cases, the terminal end of the conidia was smooth. The teleomorph was not found. Molecular and phylogenetic analysis of the ITS rDNA (2) region showed that samples are closely related to specimens of Pseudoidium anacardii (1) (teleomorph: Erysiphe quercicola [4]) collected from mango trees in diverse countries. Measurements of somatic and asexual structures are in agreement with descriptions of P. anachardii (formerly known as Oidium mangiferae) from India (3). The nucleotide sequences derived from this research were deposited in GenBank (Accession Nos. JX893951 to JX893957). To our knowledge, this is the first report of P. anacardii associated to mango inflorescences in Sinaloa, Mexico. Due to the economic importance of powdery mildew of mango trees in Sinaloa, future research directions should focus on finding the teleomorph of the fungus to support its identity. References: (1) U. Braun and R. T. A. Cook CBS Biodiversity Series No. 11, 2012. (2) S. Limkaisang et al. Mycoscience 47:327, 2006. (3) O. Prakash and K. C. Srivastava. Mango diseases and their management. A World Review Today and Tomorrow Publishers. New Delhi, India, 1987. (4) S. Takamatsu et al. Mycol. Res. 111:809, 2007.

A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorrhizal fungus Glomus intraradices is regulated in response to phosphate in the environment.

The majority of vascular flowering plants are able to form symbiotic associations with arbuscular mycorrhizal fungi. These symbioses, termed arbuscular mycorrhizas, are mutually beneficial, and the fungus delivers phosphate to the plant while receiving carbon. In these symbioses, phosphate uptake by the arbuscular mycorrhizal fungus is the first step in the process of phosphate transport to the plant. Previously, we cloned a phosphate transporter gene involved in this process. Here, we analyze the expression and regulation of a phosphate transporter gene (GiPT) in the extra-radical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices during mycorrhizal association with carrot or Medicago truncatula roots. These analyses reveal that GiPT expression is regulated in response to phosphate concentrations in the environment surrounding the extra-radical hyphae and modulated by the overall phosphate status of the mycorrhiza. Phosphate concentrations, typical of those found in the soil solution, result in expression of GiPT. These data imply that G. intraradices can perceive phosphate levels in the external environment but also suggest the presence of an internal phosphate sensing mechanism.

Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium.

Two rapid and simple in planta transformation methods have been developed for the model legume Medicago truncatula. The first approach is based on a method developed for transformation of Arabidopsis thaliana and involves infiltration of flowering plants with a suspension of Agrobacterium. The second method involves infiltration of young seedlings with Agrobacterium. In both cases a proportion of the progeny of the infiltrated plants is transformed. The transformation frequency ranges from 4.7 to 76% for the flower infiltration method, and from 2.9 to 27.6% for the seedling infiltration method. Both procedures resulted in a mixture of independent transformants and sibling transformants. The transformants were genetically stable, and analysis of the T2 generation indicates that the transgenes are inherited in a Mendelian fashion. These transformation systems will increase the utility of M. truncatula as a model system and enable large-scale insertional mutagenesis. T-DNA tagging and the many adaptations of this approach provide a wide range of opportunities for the analysis of the unique aspects of legumes.

Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme.

Many terrestrial plant species are able to form symbiotic associations with arbuscular mycorrhizal fungi. Here we have identified three cDNA clones representing genes whose expression is induced during the arbuscular mycorrhizal symbiosis formed between Medicago truncatula and an arbuscular mycorrhizal fungus, Glomus versiforme. The three clones represent M. truncatula genes and encode novel proteins: a xyloglucan endotransglycosylase-related protein, a putative arabinogalactan protein (AGP), and a putative homologue of the mammalian p110 subunit of initiation factor 3 (eIF3). These genes show little or no expression in M. truncatula roots prior to formation of the symbiosis and are significantly induced following colonization by G. versiforme. The genes are not induced in roots in response to increases in phosphate. This suggests that induction of expression during the symbiosis is due to the interaction with the fungus and is not a secondary effect of improved phosphate nutrition. In situ hybridization revealed that the putative AGP is expressed specifically in cortical cells containing arbuscules. The identification of two mycorrhiza-induced genes encoding proteins predicted to be involved in cell wall structure is consistent with previous electron microscopy data that indicated major alterations in the extracellular matrix of the cortical cells following colonization by mycorrhizal fungi.

Molecular characterization of three differentially expressed members of the Camptotheca acuminata 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) gene family.

Camptotheca acuminata is a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) supplies mevalonate for the terpenoid moiety of CPT and its hydroxylated derivative 10-hydroxycamptothecin (10-OH-CPT). We previously described the isolation of a gene encoding HMGR from C. acuminata (hmg1) and analyzed its expression in transgenic tobacco [6]. Here, we report on the isolation of genomic (hmg2) and cDNA (hmg3) clones representing two additional HMGR gene family members and characterize the expression of all three genes in C. acuminata. Transcript levels for two family members were highest in the shoot apex, dry seeds (hmg1), and bark (hmg3) which are the tissues containing the highest levels of CPT and 10-OH-CPT respectively. Levels of hmg3 mRNA also correlated with the accumulation of 10-OH-CPT during germination. In C. acuminata leaf disks, hmg1 mRNA increased in response to wounding, and this induction was suppressed by methyl jasmonate (MeJA), in agreement with results previously obtained in transgenic tobacco [6]. In contrast, wounding and MeJA did not affect hmg2 or hmg3 transcript levels in C. acuminata. These results show that members of the C. acuminata HMGR gene family are differentially expressed in various tissues under different physiological conditions which may contribute to the regulation of monoterpenoid indole alkaloid synthesis in this species.

Molecular characterization of the AP19 gene family in Arabidopsis thaliana: components of the Golgi AP-1 clathrin assembly protein complex.

AP19 is the smallest polypeptide component of AP-1, the clathrin associated protein complex found in clathrin-coated vesicles of the Golgi apparatus. Two genomic clones that encode homologues of AP19 were isolated from Arabidopsis thaliana (AAP19-1 and AAP19-2). Analysis of their nucleotide sequences predict proteins of 162 and 163 amino acids with mr of 18,913 and 18,758 respectively. Amino acid sequence comparisons with mammalian, yeast and plant clathrin associated sequences indicates that the Arabidopsis genes encode polypeptides that are more closely related to the AP19 proteins associated with clathrin-coated Golgi vesicles than to AP17, which is part of the AP-2 complex of endocytic clathrin-coated pits. Ribonuclease protection assays showed that both genes are expressed in all Arabidopsis tissues throughout development. Constitutive transcription of AAP19-1 was confirmed in transgenic Arabidopsis seedlings and plants containing an AAP19-1 promoter::beta-glucuronidase (GUS) fusion by ribonuclease protection assays and GUS histochemical staining.

Cloning and expression of a plant homologue of the small subunit of the Golgi-associated clathrin assembly protein AP19 from Camptotheca acuminata.

Clathrin-coated vesicles (CCVs) are involved in selective protein transport in eukaryotes. AP-1 and AP-2 are protein complexes found in the CCVs of the Golgi apparatus and the plasma membrane respectively. AP19 is the smallest polypeptide chain components of AP-1. We have identified a cDNA clone (CAP19) encoding a putative homologue for the assembly protein AP19 from the Chinese medicinal tree, Camptotheca acuminata. The deduced polypeptide contains 161 amino acids and has a predicted Mr of 18820. DNA blot analysis suggests that the AP19S of C. acuminata are encoded by a small gene family. CAP19 was expressed ubiquitously throughout the plant suggesting that it may be involved in general Golgi-mediated secretion.

Molecular analysis of a new member of the opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylase gene family.

An aromatic amino acid decarboxylase DNA fragment was generated from opium poppy (Papaver somniferum L.) genomic DNA by the PCR using primers designed from conserved amino acid sequences of other aromatic amino acid decarboxylase genes. Using this fragment as a probe, a genomic clone was isolated that encodes a new member of the opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylase gene family (TyDC5). The predicted TyDC5 amino acid sequence shares extensive identity with other opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylases (84%), and when expressed in Escherichia coli, it is active against tyrosine and to a lesser extent against 3,4-dihydroxyphenylalanine. Ribonuclease protection assays indicate that TyDC5 is expressed primarily in the roots of mature poppy plants. A peak of TyDC5 expression was also observed during germination, coincident with the emergence of the radicle from the seed coat. Parallel results were obtained in transgenic tobacco using a TyDC5 promoter fragment (-2060) translationally fused to the beta-glucuronidase reporter gene (GUS). IN TyDC5::GUS tobacco, GUS activity transiently appeared in all parts of the seedling during germination, but was limited to the roots in older plants. These results indicate that TyDC5 expression is transcriptionally regulated and suggest that the TyDC5 enzyme may play an important role in providing precursors for alkaloid synthesis in the roots and germinating seedlings of opium poppy.

Expression of a 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Camptotheca acuminata is differentially regulated by wounding and methyl jasmonate.

We have isolated a gene, hmg1, for 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) from Camptotheca acuminata, a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). HMGR supplies mevalonate for the synthesis of the terpenoid component of CPT as well as for the formation of many other primary and secondary metabolites. In Camptotheca, hmg1 transcripts were detected only in young seedlings and not in vegetative organs of older plants. Regulation of the hmg1 promoter was studied in transgenic tobacco using three translational fusions (-1678, -1107, -165) with the beta-glucuronidase (GUS) reporter gene. Histochemical analysis of plants containing each of the three promoter fusions showed similar developmental and spatial expression patterns. In vegetative tissues, GUS staining was localized to the epidermis of young leaves and stems, particularly in glandular trichomes. Roots showed intense staining in the cortical tissues in the elongation zone and light staining in the cortex of mature roots. hmg1::GUS expression was also observed in sepals, petals, pistils, and stamens of developing flowers, with darkest staining in the ovary wall, ovules, stigmas, and pollen. Leaf discs from plants containing each of the translational fusions showed a 15- to 20-fold wound induction of hmg1::GUS expression over 72 h; however, this increase in GUS activity was completely suppressed by treatment with methyl jasmonate. Taken together, these data show that a 165-bp fragment of Camptotheca hmg1 promoter is sufficient to confer developmental regulation as well as wound induction and methyl jasmonate suppression of GUS expression in transgenic tobacco.

Green Roots: Photosynthesis and Photoautotrophy in an Underground Plant Organ.

The potential for photosynthetic and photoautotrophic growth was studied in hairy root cultures of Asteraceae and Solanaceae species. Upon transfer to light, initially heterotrophic root cultures of Acmella oppositifolia and Datura innoxia greened rapidly, differentiated chloroplasts, and developed light-dependent CO2 fixation in the cortical cells. Photosynthetic potential was expressed in root cultures of all the Asteraceae genera examined (Acmella, Artemisia, Rudbeckia, Stevia, and Tagetes). Hairy roots of A. oppositifolia and D. innoxia were further adapted to photoautotrophy by growing in the presence of light and added CO2 (1-5%) and by direct or sequential transfers into media containing progressively lower sugar concentrations. The transition to photoautotrophy was accompanied by an increase in CO2 fixation and in the specific activity of 1,5-ribulose-bisphosphate carboxylase/ oxygenase (Rubisco). During the adaptation of A. oppositifolia roots to photoautotrophy, the ratio of Rubisco to phosphoenolpyruvate carboxylase increased significantly, approaching that found in the leaves. The levels and patterns of alkaloids and polyacetylenes produced by Solanaceae and Asteraceae hairy roots, respectively, were dramatically altered in photomixotrophic and photoautotrophic cultures. Photoautotrophic roots of A. oppositifolia have been mainitained in vitro for over 2 years.