Global tuberculosis research and its future prospects.

Tuberculosis is the infectious disease that causes the most deaths each year in the world. Around 25% of the population is estimated to be infected with, Mycobacterium tuberculosis, the bacteria that gives rise to the disease, and more than one and a half million people die each year from this cause. A rigorous bibliometric analysis has been developed around tuberculosis disease, and the most remarkable results are presented in this paper. It is observed that interest in tuberculosis is growing, and the control of its spread has become one of the main health priorities in the world, with the United States, the United Kingdom, and India, leading the research in this area. On the other hand, it has been observed that there are two main health concerns around the tuberculosis: drug-resistant tuberculosis and co-infection with HIV. Finally, conclusions are offered, playing a frontline role in science policy decisions and research performance evaluations.

Practical approach to the evaluation of industrial wastewater treatment by the application of advanced microbiological techniques.

In cork industry, the operation of boiling raw cork generates large volumes of wastewater named Cork Boiling Wastewater (CBW). The main characteristics are the low biodegradability and medium to low acute toxicity, resulting in the necessity of designing advanced biological treatments by possible conventional activated sludge adaptation. In order to evaluate the variation of bacterial population along that process, a study based on optical microscopy, plate count, DNA extraction, qPCR and massive sequencing techniques was performed. Results showed a diminution of the total and volatile solids (TSS and VSS), jointly with a decrease in DNA concentration, general bacteria (16 S) and ammonia-oxidizing bacteria (AOB). After a few hours of testing, diverse microbiological species died while others showed a possible adaptation of the biological system, accompained by a dissolved organic carbon (DOC) reduction. In addition, toxicity tests based on activated sludge showed the development of chronic toxicity through the contact time. Combination of classical and advanced microbiological techniques, such as quantitative real time Polymerase Chain Reaction (qPCR) and metagenomics, was essential to predict the variation of species during the experiment and to conclude if effective biological adaptation could be finally attained for the target complex wastewater.

First Report of Fusarium equiseti Causing Damping-Off Disease on Aleppo Pine in Algeria.

The Aleppo pine (Pinus halepensis Mill.) is a conifer native to the Mediterranean region. In 2008 and 2009, a survey of Aleppo pine seedling diseases was performed in three forest nurseries from Relizane, Sidi Bel Abbes, and Tlemcen provinces in northwestern Algeria. Aleppo pine seedlings showed symptoms of pre- and post-emergence damping-off disease, with an incidence of 64 to 77%. Four composite samples were taken from each location. Disinfested root and root collar segments, approximately 5 mm in length, were cultured on potato dextrose agar (PDA) and incubated at 25°C, and hyphal tips were transferred to PDA. Fusarium equiseti (Corda) Sacc. (teleomorph: Gibberella intricans Wollenw.) was identified from roots of two seedlings from the Sidi Bel Abbes nursery. Morphological identification was done according to Fusarium keys (2). PDA colonies with abundant, loosely floccose, whitish aerial mycelium and beige pigmentation were observed. Macroconidia with usually 5 to 6 septa, 31 to 45 µm long. A pronounced dorsiventral curvature, tapered and elongated apical cell, and prominent foot shape were observed. Microconidia were absent. Chlamydospores were produced in hyphae, most often intercalary, solitary, in pairs, frequently forming chains or clusters, globose (7 to 13 µm). To confirm the identity of this fungus, the internal transcribed spacer of F3RS1 and F19RS1 isolates of F. equiseti were amplified and sequenced using ITS1 and ITS4 primers (4), GenBank accession nos. JX114784 and JX114791, respectively. Those sequences bore 100% (HQ671182) similarity with sequences of F. equiseti in GenBank. Pathogenicity tests were performed to fulfill Koch's postulates. Inoculum was produced by adding a 5-mm-diameter plug from a 7-day-old CMA petri dish culture to a previously sterilized 500 ml flask (237.5 g sand, 12.5 g cornmeal, 80 ml sterile distilled water), shaken over 9 days at 25°C, and mixed with sterile sandy clay soil at 1:3 (v:v). Infested soil was then transferred to 500 ml pots, and 10 Aleppo pine seeds were planted per pot. A completely randomized design was used with three replicates per isolate and three control pots with a similar non-infested soil. After 1 month at 25°C the two tested isolates caused typical damping-off symptoms (collar rot) on seedlings and were re-isolated from recently infected tissues. The percentages of the inoculated plants that became infected were 59 to 65% among isolates (0% in control pots). To our knowledge, infection by F. equiseti is a first report on Aleppo pine in northwestern Algeria, Northern Africa, and globally, and on conifers in the Mediterranean region (1,3). In Algeria, F. equiseti is associated with black pepper (Piper nigrum L.) (3). These findings highlight the moderate impact of F. equiseti on the production of Aleppo seedling stock for reforestation activities in northwestern Algeria. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory. ARS, USDA, Beltsville, MD. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , February 20, 2013. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) D. W. Minter. Cybertruffle's Robigalia, Observations of Fungi and their Associated Organisms. Retrieved from http://www.cybertruffle.org.uk/robigalia/eng/ , February 20, 2013. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Globisporangium ultimum Causing Pythium Damping-Off on Aleppo Pine in Algeria, Africa, and the Mediterranean Region.

Globisporangium ultimum (Trow) Uzuhashi, Tojo & Kakish. (syn. Pythium ultimum Trow, syn. P. ultimum Trow var. ultimum) is a known oomycetal species from Pythium s.l. causing damping-off and/or root rot on a great variety of plants throughout the world, including some pine species (Pinus L.) and conifers (2,3). Aleppo pine (Pinus halepensis Mill.) is a common native forest tree in the Mediterranean region. Pre- and post-emergence damping-off disease symptoms were observed during 2008 and 2009 in four forest nurseries from northwestern Algeria (Relizane, Sidi Belabes, and Tlemcen departments). This disease occurred under cool conditions, and Aleppo pines were significantly affected, reducing seedling emergence. Disinfected segments, about 5 mm in length, from decayed root and collar, were cultured on CMA at 25°C. This oomycetal species was identified based on the species description in Pythium keys (3,4). For the molecular identification, PCR was used to amplify the ITS region of Pythium isolates. It was amplified with the flanking primers ITS1 and ITS4, and these products were directly sequenced. Sequence data were compared to known sequences deposited in the NCBI non redundant database to confirm morphological identification. A BLAST search identified U3CR, U7CR, U1RT, U2CR, U4CR, U14CR, U7RT, and U17RT isolates (GenBank Accession Nos. JX191921, 22, 27, 29, 31, and 33 to 35, respectively) as G. ultimum based on 100% similarity with corresponding sequence of the reference isolate no. UZ056 MAFF240024 (AB468781) (3). Phytopathogenicity testing was conducted in a petri dish and pot experiment. In the petri dish experiment, a 3 mm diameter plug was transferred from a 7-day-old CMA colony to the center of a CMA petri dish, with three replicates per isolate, and three control plates were inoculated with sterile agar plugs. After 72 h, 10 Aleppo pine seeds were placed equally spaced to 1 cm from the edge of each plug. After 7 days at 22°C in the dark, germination inhibition (46.1 to 87.6%) and root growth inhibition (62.3 to 92.2%) were calculated. In the control plates, germination failure (13.4%) and root length (27.7 cm) were observed. For the pot experiment, inocula were produced by adding a 5 mm diameter plug from a 7-day-old CMA culture to a previously sterilized 500 ml flask containing 237.5 g sand, 12.5 g cornmeal, and 80 ml SDW. Nine-day-old inoculum was mixed with sterile soil at a rate of 1:3 (v:v). Inoculum was transferred to 500 ml pot, and 10 Aleppo pine seeds were planted, with three replicates per isolate, and three control pots were used. After 2 weeks, all of the isolates tested caused typical symptoms of Aleppo pine Pythium damping-off, the percentage of inoculated plants that became infected was 36.6 to 83.3%. In the control pots, no infected plants were observed. To our knowledge, this is the first report of G. ultimum causing damping-off on Aleppo pine in Algeria, Africa, and the Mediterranean Region. Before, Aleppo pine damping-off caused by G. ultimum was reported in Australia (1). References: (1) R. P. Cook and A. J. Dubé. Host-pathogen index of plant diseases in South Australia. SADA, Melbourne, Australia, 1989. (2) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory. ARS, USDA, Bestville, MD. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , June 24, 2012. (3) S. Uzuhashi et al. Mycoscience 51:337, 2010. (4) A. J. van der Plaats-Niterink. Stud. Mycol. 21:1, 1981.

First Report of Fusarium redolens as a Causal Agent of Aleppo Pine Damping-Off in Algeria.

Aleppo pine (Pinus halepensis Mill.) is a common native coniferous tree in the natural forests of the Mediterranean region. In 2008 and 2009, a survey of Aleppo pine seedling diseases was performed in three forest nurseries from Relizane, Sidi Bel Abbes, and Tlemcen departments in northwestern Algeria. Aleppo pine seedlings showed symptoms of pre- and post-emergence damping-off, resulting in severe crop losses. The problem was widespread with a disease incidence of 64 to 77%. Fusarium redolens Wollenw. was isolated from Relizane and Sidi Bel Abbes forest nurseries. Disinfested root and root collar segments, approximately 5 mm in length, were cultured on potato dextrose agar (PDA) and incubated at 25°C. Morphological identification was done according to Fusarium keys (2). PDA colonies consisted of flat mycelium with sparse white aerial hyphae. Macroconidia with three to five septa, 24 to 43.8 µm long, widest upper third, hooked apical cell, and foot shaped basal cell were observed. Microconidia with zero to one septa, 6.8 to 10.4 µm long, oval to cylindrical, and produced on monophialides were also observed. Chlamydospores were produced abundantly in terminal and intercalary chains, in 3- to 4-week-old cultures. To confirm the identity of the fungus, the internal transcribed spacer (ITS) of F5RS3, F91SR, F55RS1, F8RS3, and F09SS1 isolates of F. redolens were amplified and sequenced using ITS 1 and ITS 4 primers (3). GenBank Accession Nos. are JX051323 to 26, and JX114783, respectively. Those sequences bore 99% (JF311916) and 100% (U34565) similarity with sequences of F. redolens in GenBank. A Fusarium pathogenicity assay was used to complete Koch's postulates. Inoculum was produced by adding a 5 mm diam. agar disc from a 7-day-old CMA petri dish culture to a previously sterilized 500 ml flask (237.5 g of sand, 12.5 g of cornmeal, and 80 ml of deionized H2O). Isolates were allowed to colonize the medium for 9 days, and flasks were shaken every day. The inoculum was mixed with sterile soil at a rate of 3:1 (v:v). Infested soil was then transferred to 500 ml pots, and 10 Aleppo pine seeds were planted. A completely randomized design was used with three replicates. After 1 month, all tested isolates caused typical damping-off symptoms on seedlings. The percentage of the inoculated plants that became infected was 53 to 91%. To our knowledge, this is the first report of F. redolens being pathogenic on Aleppo pine in northwestern Algeria and throughout the world. In Algeria, F. redolens has been reported on tomato (Solanum lycopersicum L.) (1), suggesting that it is adapted to the conditions of this area and could become a major threat to regional plant production. The annual economic impact of this disease was estimated at approximately US$50,000 per forest nursery. References: (1) N. Hamini-Kadar et al. New Dis. Rep. 22:3, 2010. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, Iowa, 2006. (3) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Fusarium chlamydosporum Causing Damping-Off Disease on Aleppo Pine in Algeria.

The Aleppo pine (Pinus halepensis Mill.) is a conifer native to the Mediterranean region. In 2008 and 2009, a survey of Aleppo pine seedling diseases was performed in three forest nurseries from the Relizane, Sidi Bel Abbes, and Tlemcen departments in northwestern Algeria. One- to two-month-old Aleppo pine seedlings showed symptoms of damping-off in pre- and post-emergence (typical seedling collar rot). The problem was widespread with a disease incidence of 64 to 77% and an annual impact of US$50,000. Disinfested root and root collar segments (from four composite samples per location), approximately 5 mm in length, were cultured on PDA and incubated at 25°C and day/night light. Two (from 21) isolates were identified morphologically (2) as the anamorph Fusarium chlamydosporum Wollenw. & Reinking and isolated from collar rots of Relizane forest nursery seedlings. Colony development on PDA media was fast; 32 mm diameter colonies developed after 3 days. Colonies were white. Mycelia were floccose, fairly dense, off-white, and turned a lilac color in older portions of the colony. Macroconidia were thick-walled and moderately curved with unequal dorsiventral curvature (the lower wall is almost straight), short, curved and pointed apical cell, usually notched, but occasionally foot shaped basal cell, 3- to 5-septate, and 2 × 8 to 21 µm. Microconidia were abundant, 0-septate, and 2 × 6 to 9 µm. Chlamydospores were abundant, formed rapidly in single chains or clusters, and 8 to 15 µm diameter. To confirm the identity of this fungus, the internal transcribed spacer of F12RR and F4SR isolates of F. chlamydosporum were amplified and sequenced using ITS1 and ITS4 primers (4). Sequences were deposited in GenBank under accessions JX114795 and JX114789, respectively. Those sequences bore 99% similarity with reference sequence AY213655 (2) and 100% with HQ671187, also found 99 to 100% similarity with F. equiseti (Corda) Sacc. but with different conidia. Pathogenicity tests were performed to fulfill Koch's postulates. Inoculum was produced by adding a 5 mm diam. plug from a 7-day-old CMA petri dish culture to a previously sterilized 500 ml flask (237.5 g sand, 12.5 g cornmeal, 80 ml SDW), shaken over 9 days, and mixed with sterile soil at 1:3 (v:v). Infested soil was then transferred to 500 ml pots, and 10 seeds were planted. A completely randomized design was used with three replicates per isolate and three control pots. After 1 month, two tested isolates caused typical damping-off symptoms on seedlings. The percentage of the plants that became infected was 65 to 77%. To our knowledge (1,3), this is the first report of F. chlamydosporum on Aleppo pine in northwestern Algeria. It is also the first report of this fungal species affecting the Aleppo pine throughout the world, and on conifers in Africa and the Mediterranean region (1,3). References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Syst. Mycol. Microbiol. Lab. ARS, USDA, Beltsville, MD. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , February 20, 2013. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) D. W. Minter. Cybertruffle's Robigalia, Observations of Fungi and their Associated Organisms. Retrieved from http://www.cybertruffle.org.uk/robigalia/eng/ , February 20, 2013. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Fusarium acuminatum Causing Damping-Off Disease on Aleppo Pine in Algeria.

The Aleppo pine (Pinus halepensis Mill.) is a conifer native to the Mediterranean region. In autumn and spring of 2008 to 2009, a survey of Aleppo pine seedling diseases was carried out in three forest nurseries from the Relizane, Sidi Bel Abbes, and Tlemcen departments in northwestern Algeria. Aleppo pine seedlings were potted from the soil. In all three nurseries, 1- to 2-month old seedlings showed symptoms of damping-off disease in pre- and post-emergence (collar rot) with a disease incidence of 64, 77, and 72%, respectively. Disinfected collar segments, about 5 mm in length, were plated on PDA and petri dishes incubated at 25°C. A Fusarium sp. was consistently isolated from tissues and all isolates were morphologically identified as Fusarium acuminatum Ellis & Everh. (teleomorph: Gibberella acuminata Wollenw.) according to Fusarium keys (2). Colony growth was 43 mm after 3 days on PDA; the aerial mycelium was white, developing a brownish tinge in the center on PDA; macroconidia were formed in orange sporodochia, broadly falcate, strongly septate, 3 to 5 septa, the apical cell with an incurved elongation, distinct foot shape, 3 to 4 × 20 to 50 µm; microconidia were usually absent for isolates other than F12SS1, reniform, septate, 5 to 6 × 6 to 10 µm, in monophialides; chlamydospores were formed in chains, 6 to 13 µm. For the molecular identification, ITS regions of Fusarium isolates were amplified with the primers ITS1 and ITS4, and products were directly sequenced in both strands using the same primers ITS 1 and ITS4. Sequences were compared to known sequences deposited in the NCBI non redundant database to confirm morphological identification. An NCBI BLAST search identified isolates F12SS1, F14SS3, F30SS3, and F25SR as F. acuminatum based on 100% similarity with corresponding sequences. GenBank Accession Nos. were JX114788, JX114785, JX114782, and JX114790, respectively. Pathogenicity tests were performed to fulfill Koch's postulates. Inocula were produced by adding a 5-mm diameter plug from a 7-day-old CMA petri dish culture to a previously sterilized 500-ml flask (237.5 g sand, 12.5 g cornmeal, 80 ml SDW), shaken over 9 days, and mixed with sterile soil at 1:3 (v:v). The inocula were transferred to a 500-ml pot, and 10 Aleppo pine seeds were planted with three replicates. After 1 month, all tested isolates caused typical symptoms on seedlings and the proportion of infected seedlings per each isolate was 50, 53.33, 56.66, 60, and 63.33%, respectively. There are many reports of F. acuminatum associated to conifer seedlings in nurseries (1,3) and most of them are conflicting because in some reports this species is considered non-pathogenic or only a seed contaminant and others consider it as a pathogen. To our knowledge, F. acuminatum is a first report on the Aleppo pine in northwestern Algeria, northern Africa. It is also the first report of this fungal species affecting the Aleppo pine throughout the world, and on conifers in Africa and the Mediterranean region. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory. ARS, USDA., Bestville, Maryland, USA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , June 18, 2012. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, Iowa, USA, 2006. (3) D. W. Minter. Cybertruffle's Robigalia, Observations of Fungi and their Associated Organisms. Retrieved from http://www.cybertruffle.org.uk/robigalia/eng/ , June 18, 2012.

Cloning and molecular characterization of the acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1) gene from Echium.

Boraginaceae species, such as those from the genus Echium, contain high levels of the Delta(6)-desaturated gamma-linolenic (18:3n-6) and octadecatetraenoic (18:4n-3) acids. These are unusual fatty acids among the plant kingdom that are gaining interest due to their benefits to human health. The potential utility of acyltransferases aimed at an increase in oil yield and fatty acid profiling has been reported. In this work, a gene encoding an acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) was cloned from Echium pitardii. Genomic and cDNA sequences obtained revealed a gene structure composed of 16 exons, yielding a protein (EpDGAT) of 473 amino acids with high similarity to DGAT1 enzymes of plants. Protein features such as a predicted structure with a highly hydrophilic N-terminus followed by 10 transmembrane domains, as well as the presence of diverse specific signatures, also indicate that EpDGAT belongs to the DGAT1 family. indeed. DGAT activity of the protein encoded by EpDGAT was confirmed by heterologous expression of the full-length cDNA in a yeast mutant (H1246) defective in the synthesis of triacylglycerols. Fatty acid composition of the triacylglycerols synthesized by EpDGAT in H1246 yeast cultures supplemented with polyunsaturated fatty acids suggest a substrate preference for the trienoic fatty acids alpha-linolenic acid (18:3n-3) and gamma-linolenic acid over the dienoic linoleic acid (18:2n-6). Site-directed mutagenesis has revealed the presence of a critical residue (P(178) in EpDGAT) within a reported thiolase signature for binding of acyl-enzyme intermediates that might be involved in the active site of the enzyme. Transcript analysis for EpDGAT shows an ubiquitous expression of the gene which is increased in leaves during senescence.