David Henry Moon

Comparative Analyses of the Complete Genome Sequences of Pierce's Disease and Citrus Variegated Chlorosis Strains of Xylella fastidiosa

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X. fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierces disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X. fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X. fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X. fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.

Miniprep DNA isolation from unicellular and filamentous cyanobacteria.

A rapid miniprep method for isolation of DNA from 12 strains of cyanobacteria belonging to groups I, III, IV and V is described. The protocol is a modification of the methods of Boyle and Lew [Boyle, J.S., Lew, A.M., 1995. An inexpensive alternative to glassmilk for DNA purification. Trends Genet. 11, 8] and the cetyltrimethyl ammonium bromide (CTAB) extraction method of Sahgai-Maroof et al. [Sahgai-Maroof, M.A., Soliman, K.M., Jorgensen, R.A., Allard, R.W., 1984. Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics. Proc. Natl. Acad. Sci. USA 81, 8014-80181. The new method is especially useful for obtaining cyanobacterial DNA from unicellular, filamentous and filamentous branched species. The method does not require phenol extraction and the product can be used directly for PCR amplification and restriction digestion.

Algicide production by the filamentous cyanobacterium Fischerella sp. CENA 19

The biosynthesis of algicides produced by a novelFischerellastrain was investigated. Two allelochemicals were identified, the aminoacylpolyketide fischerellin A (FsA) and the alkaloid 12-epi-hapalindole F (HapF). Based on the structure of FsA, genes that could be involved in its biosynthesis, including those encoding nonribosomal peptide synthetases (NRPSs) and a polyketide synthase (PKS), were identified by the polymerase chain reaction (PCR). By showing that the expression of NRPSs and PKSs is concomitant with algicide production we suggest that the identified genes may be involved in algicide biosynthesis. Analysis of an algicide preparation of the Brazilian-Amazonian strainFischerellasp. CENA 19 revealed the production of FsA,m/z409 (MH+), HapF,m/z370 (MH+), and other potential isoforms of the latter compounds, which were identified by high-performance liquid chromatography (HPLC) and matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass-spectrometry. The production of HapF was confirmed after purification by HPLC, analysis by NMR, and high-resolution mass-spectrometry (HRMS). Two-NRPS and a PKS gene were identified after specific amplification using a degenerate PCR. The expression of these synthetases was confirmed by Western blot analysis employing enzyme family-specific antibodies. These analyses revealed the presence of three NRPSs and a single PKS inFischerellasp. CENA 19. The structure of FsA indicates both aminoacyl- and polyketide moeities, suggesting that its biosynthesis may require an integrated NRPS/PKS enzyme system, possibly involving the genes and the synthetases identified.

An alternative method for Staphylococcus aureus DNA isolation

This study describes a rapid procedure for the isolation of genomic DNA from Staphylococcus aureus that yielded a good amount of high quality DNA for the amplification of staphylococcal enterotoxins genes (A, B, C, D, and E) and the TSST-1 gene as well as enzymatic restriction (HaeIII) from environmental isolates. With this method, it was possible to detect these genes in a sample containing as little as 105 cells with positive PCR reactions obtained from approximately 10pg of DNA in a final reaction volume of 25µl.

Effects of host plant origin on nodulin activities and nitrogen fixation in Phaseolus vulgaris L.

The metabolic activities of root nodules formed by Rhizobium tropici UM1899 were measured to test for the effects of geographical origin of the host bean (Phaseolus vulgaris L.) plant. Under increasing levels of N (0 to 24 mM of NH4NO3), the optimum condition for nitrogen fixation based on nitrogenase activity and allantoin concentration, was obtained between 2 and 4 mM N. Cultivars, including wild accessions from the two major domestication centers in America (Middle America and Andes), were then grown under aseptic conditions with 2 mM NH4NO3 and the rhizobial inoculant. Plant nodulins [leghaemoglobin (Lb), phosphoenolypyruvate carboxylase (PEPC) and glutamine synthetase (GS)], bacterial nitrogenase (NIF) activities as well as allantoin (ALA) concentration in the xylem sap, were assayed in flowering plants. Lb, PEPC, NIF activities and ALA concentrations were strongly affected by cultivar and by the center of origin. GS activity did not vary significantly with either cultivar or center of origin. LB, NIF and ALA were directly related to plant growth and offer opportunities to select for efficient N2-fixing symbioses. There were slight increases in nodulin activities of the domesticated cultivars, but the overall low variability within this material relative to landraces suggests that diversity for biological nitrogen fixation was reduced by domestication.

Genetic Variability in Puccinia psidii Populations as Revealed by PCR-DGGE and T-RFLP Markers

Eucalyptus rust caused by Puccinia psidii is responsible for losses of approximately 20% of young Eucalyptus plants, depending on the environmental conditions and the geographic location. Despite its economic importance, there are few studies describing the genetic variability in P. psidii populations that infect different host plants. In the present study, we evaluated the ribosomal DNA internal transcribed spacer region (rDNA-ITS) using polymerase chain reaction denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism to assess the genetic variability in P. psidii populations infecting different Eucalyptus spp. and hybrids, as well as guava, jabuticaba, and syzygium. These culture-independent methods were efficient in differentiating populations based on the host species from which they were collected. In general, the results from both techniques showed that the populations collected from guava, jabuticaba, and syzygium were different from and had a greater level of diversity than the Eucalyptus rust populations. The sequencing of cloned rDNA-ITS fragments confirmed that the vast majority of the profiles generated were from P. psidii. This analysis also revealed interesting single-nucleotide polymorphisms. Therefore, these culture-independent methods are suitable for the rapid assessment of genetic variability within and between populations of this biotrophic fungus on a variety of host species and could be a tool to study the evolution of this pathogen and its interactions with host plants.

Cloning and endogenous expression of a Eucalyptus grandis UDP-glucose dehydrogenase cDNA

UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis.

A comparison of two radiometric methods to investigate the biodegradation of phenol by Candida nitrativorans

Abstract Traditional radiometric methods use internal CO 2 collectors to follow the degradation process of a radiolabeled compound by the release of 14 CO 2 in an essentially closed system that is opened repeatedly for sampling. Our aim was to investigate the biodegradation of phenol by C . nitrativorans using radiolabeled phenol in internal and external collection systems. Using the internal system, the loss of activity from the control vessel was approximately 26% and in the experimental vessel, losses reached 57%. We attribute these losses to phenol volatilization (control) and volatile products from degradation (experimental vessel), during the sampling process. An external collector system using forced air flow, was devised that allowed the collection of the 14 CO 2 produced in a series of three external collectors. The losses observed using this system were greatly reduced, 32% in the experimental vessel, with the uninoculated control accounting for 25%. Therefore, only 7% of the initial activity was lost or unaccounted for. It was also shown that the activity collected from the control vessel was not due to non-biological degradation of the phenol producing CO 2 , but rather to the volatilization of the phenol. Comparing the two methods, it was noted that the external collection system recovered more of the total initial activity than did the internal system and less activity was detected in the control flask, indicating lower losses through volatilization of the phenol substrate. Extrapolating the data from the control in the external system after precipitation with BaCl 2 , we would suggest that the activity in the internal collector system control was due to the capture of phenol and not to the capture of CO 2 . Based on these data, we would recommend the use of the external collection system for radiorespirometric measurement of an aerobic degradation process, using a substrate which is sensitive to the disruption of the gas/liquid phase equilibrium.

A simple protocol for whole leaf preparation to investigate the interaction between Puccinia psidii and Eucalyptus grandis

During the infection of a susceptible Eucalyptus tree by the biotrophic fungus Puccinia psidii a series of processes occur, starting with germination of the fungal spore, through hyphal penetration, colonization and finally sporulation. Not all of these processes occur when the fungus tries to infect a resistant Eucalyptus tree. Such interactions need to be studied to allow future molecular analyses to be correlated with the physical/biological state of the infection in both resistant and susceptible hosts. In this manuscript we describe a simple protocol that allowed us to follow the infection cycle, both on the surface and within the leaf tissue, of Puccinia psidii on its host Eucalyptus grandis. The procedure consists of fixing the tissue with ethanol and acetic acid, clearing with hot KOH and staining with calcofluor before fluorescence detection. All of the previously mentioned infection steps, including pustule rupture and spore release, were observed. Germ tubes, appressoria, haustorial mother cells and urediniospores were all clearly seen. This protocol also proved useful in tissue preparation for scanning electron microscopy and confocal microscopy.