Transcriptome sequencing and analysis of the coconut leaf beetle, Brontispa longissima.

The coconut leaf beetle, Brontispa longissima, is a destructive pest of palm plants. Although its ecological and biological characteristics are well understood, its genetic information remains largely unknown. To advance our understanding of its molecular ba-sis of biology and ecology, we sequenced and analyzed its whole transcriptome by using high-throughput Illumina paired-end sequencing technology. Approximately 8.08 Gb of clean reads were generated in a single run, which were assembled by using Trinity into 41,652 unigenes with an average length of 932 bp. By sequence similarity searches for known proteins, 23,077 (55.4%) unigenes were annotated by BLASTx searches against the NCBI non-redundant protein database. Of the unigenes assembled, 18,153 and 13,733 were assigned to Gene Ontology and Clusters of Orthologous Groups of proteins, respectively. In addition, 10,415 unigenes were mapped onto 247 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. These transcriptomic resources will facilitate gene identification and elucidate the molecular mechanisms of biological and ecological aspects under-lying this palm pest, in order to design a new control strategy.

Transcriptomic identification of chemoreceptor genes in the red palm weevil Rhynchophorus ferrugineus.

Olfaction is crucial for insects' survival because it enables them to recognize various environmental information. It is primarily mediated by a large family of chemoreceptors, including olfactory receptors (ORs), gustatory receptors (GRs), and ionotropic receptors (IRs). Here, we assembled the transcriptome of the economically important pest of palms, Rhynchophorus ferrugineus, to reveal its chemoreceptor gene repertoire. About 8.08 Gbp data were generated using a HiSeq platform and their assembly led to a total of 24,439 unigenes. Among the transcripts, 12,523 (51.24%) showed significant similarity (E-value <10(-5)) to known proteins in the National Center for Biotechnology Information Nr database. From these sequences, 18 candidate genes of ORs were identified. Nine putative transcripts were homologous to GR genes, while 9 were similar to IR genes. The expression profiles of all identified chemoreceptor genes were determined by quantitative real-time PCR in antenna, head, thorax, abdomen, and legs of both sexes. Most chemoreceptor genes were antenna-enriched. This study demonstrated a successful application of a transcriptome for discovering a large number of divergent chemoreceptor genes of a non-model organism. The findings provide a valuable sequence resource and gene tissue distribution information for systematic functional analysis of molecular mechanisms underlying chemoreception in this pest.

First Report of Pindo Palm Heart Rot Caused by Ceratocystis paradoxa in China.

On January 12th, 2012, a novel disease with an incidence of 50% was discovered in Pindo palm Butia capitata (Mart.) Becc from the Coconut Grant View Garden (19°33.137' N, 110°47.482' E) located in Wenchang, Hainan Province. Diseased leaflets at the base of the rotted heart leaves had reddish brown lesions; when the infection progressed, the leaves turned yellow and became blighted from the inner to the outer part of the crown. Once the growing point was destroyed, the entire tree ultimately died. Tissues from the edges of lesions from diseased leaflet samples were placed onto potato dextrose agar (PDA) and incubated at 25°C for 3 days. The color of colonies of five isolates obtained turned from white to black in 48 h. The optimum temperature for mycelium growth was from 20 to 30°C, and no growth occurred at temperatures higher than 40°C or lower than 5°C (n = 5). The cylindrical colorless to pale brown conidia were 7.5 to 17.5 µm long × 5.0 to 7.5 µm wide (n = 100); oval black chlamydospores were 12.5 to 22.5 × 7.5 to 15.0 µm (n = 100). The sequence (497 bp) of the internal transcribed spacer (ITS) region of the representative isolate BX3 (China Center for Type Culture Collection No. CCTCC AF2014002) was amplified using primer pair ITS1/ITS4 (GenBank Accession No. KF939052) and shared 99% sequence identity with Ceratocystis paradoxa strain xie331-4 (JQ039332). Based upon these biological characteristics and ITS sequence, this pathogen was identified as C. paradoxa (Dade) C. Moreau (anamorph Thielaviopsis paradoxa (de Seynes) Höhn.) (3). Pathogenicity tests were conducted on 8-cm-long sections of young leaflets excised from a 12-year-old pindo palm tree. One side of the midrib of 10 sections was wounded with a sterilized scalpel at the center and the other side was non-wounded, then a PDA plug (4 to 6 × 4 to 6 mm) from the edge of an actively growing colony of BX3 incubated for 3 days were inoculated onto each wounded or non-wounded site. As controls, plain PDA plugs were placed on wounded and non-wounded spots of another 10 sections following the above procedure. Pathogenicity was tested twice. Each inoculated section was then put into a 9-cm petri dish in which two filter papers (Φ = 9 cm) were placed and 8 ml of sterile water were added to maintain high humidity, and then all dishes were placed in a dark incubator at 25°C. After 5 days, typical symptoms developed only on the wounded points inoculated with mycelium plugs. C. paradoxa was re-isolated from the margins of the expanding lesions. C. paradoxa causing fruit rot of B. capitata was reported in Uruguay (2), but to our knowledge, there are no previous reports of this species in China or infecting leaves of B. capitata worldwide (1). We report here a new Ceratocystis disease on B. capitata, and it was named as pindo palm heart rot based on its symptoms. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , Feb 21, 2014. (2) V. Gepp et al. New Dis. Rep. 27:12, 2013. (3) F. Y. Yu et al. Plant Dis. 96:290, 2012.

First Report of Leaf Spot Disease in Coconut Seedling Caused by Bipolaris setariae in China.

Coconut (Cocos nucifera L.), an important oilseed as well as a multipurpose perennial plantation crop, is distributed and planted in humid tropical areas. In October 2012, a new leaf spot disease was observed on 3-year-old coconut seedlings in Wenchang, Hainan Province, China. The symptom first appeared as spindly or elliptical and brown flecks with water-soaked lesions that became yellow with the progress of the disease. In the later stage of the disease, the lesions merged together, gradually expanding to the leaf apex. In recent years, the disease has been prevalent in all the nursery gardens surveyed. Once young leaves got infected and nearly all the leaves of the tree showed diseased symptoms, the coconut eventually became defoliated. The pathogen was isolated from the lesion margin, surface sterilized with 75% ethanol and 0.1% mercury bichloride, washed by sterile distilled water, and then placed excising pieces of leaves from the leision margin onto potato dextrose agar (PDA). Plates were incubated at 25°C for 4 days. After 7 days, the colony was grayish black and produced black pigment in the medium. Aerial mycelium was fluffy, septate, and branched, the conidiophores were slightly flexuous or straight, 5 to 11 µm thick, and produced curved, spindle-shaped, or fusiform, septate conidia with 4 to 10 septa, measuring 39 to 86 × 9 to 16 µm, with a slightly protuberant hilum, truncated. Based on the symptoms and mycelial and conidial characters above, the fungus was identified as Bipolaris setariae (1). The pathogenicity was established and repeated for six times by following Koch's postulates. Two 1-year-old coconut seedlings were washed with sterilized water and six leaves were wounded with a sterile needle and then inoculated by spraying them with a suspension of conidia of the isolate. The seedlings were kept in two incubators at 25°C for 12 days. Inoculated leaves showed typical symptoms similar to those described above. The pathogen was re-isolated from inoculated leaves. Morphological characteristics were identical to the original isolated fungus. In contrast, the control leaves did not show any symptoms. The genomic DNA of this fungus was extracted, amplification of the internal transcribed spacer (ITS) region was performed with primer ITS1 and ITS4, and the purified PCR product was sequenced (GenBank Accession No. KJ605157). BLASTn analysis revealed 99% sequence similarity with four B. setariae isolates (HE792936.1, JX462256, GU073108.1, and FJ606786.1). Morphologic characters and sequence analysis of the ITS rDNA confirmed that the pathogen was B. setariae. Bipolaris incurvata has been reported causing disease on coconut (2), but B. setariae was not previously reported on coconut. So far, this is the first report of B. setariae caused coconut seedling leaf spot disease in Hainan, China. References: (1) K. C. da Cunha et al. J. Clin. Microbiol. 50:4061, 2012. (2) A. Kamalakannan et al. New Dis. Rep. 12:18, 2005.

First Report of Leaf Spot of Tea Oil Camellia (Camellia oleifera) Caused by Lasiodiplodia theobromae in China.

Tea oil camellia (Camellia oleifera Abel.), one of the most famous woody oil plants, is distributed and cultivated widely in central and southern China for its strong adaptability. In September 2013, tea oil camellia plants with severe leaf spots were observed in commercial production fields located in Wenchang, Hainan Province. Spots were initially chlorotic, became necrotic and black with a chlorotic halo, developing to cover the entire width of the leaves, and leading to leaf death. Isolations were performed by excising pieces of symptomatic leaves from the lesion margin, surface sterilized with 90% ethanol and 0.6% sodium hypochlorite, and then placed them on potato dextrose agar (PDA). Plates were incubated in a sterile chamber at 26 ± 2°C for 2 days. A fungus was consistently isolated on PDA from all 23 diseased leaf samples. Pure cultures were obtained by monosporic culture technique. After 2 to 3 days of incubation at 26 ± 2°C with a 12-h photoperiod, the fungus initially produced white colonies with dense aerial mycelia, which later turned black (6 to 7 days). The mycelium was fast spreading, branched, and septate. Pycnidia were black, globose, ostiolate, and produced in stroma on the medium surface after 28 days at the same culture conditions as above. Conidia were initially unicellular, subovoid, hyaline, thick-walled with granular content, and 19.8 to 28.9 × 11.5 to 15.7 µm (avg. 25.1 × 13.5 µm). Mature conidia were one-septate and dark brown with longitudinal striations. These observed morphological features suggested that the fungus possessed the same characteristics as previously described for Lasiodiplodia theobromae (Pat.) Griffon & Maubl (syn = Botryodiplodia theobromae) (2). For molecular identification, the ITS1-5.8S-ITS2 region and fragments of the ß-tubulin and elongation factor 1-alpha (EF1-α) genes were sequenced and BLASTn searches done in GenBank. Accession numbers of gene sequences submitted to GenBank were KF811055 for ITS region; KJ639047 for ß-tubulin; and KJ639048 for EF1-α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS164.96 of L. theobromae reported in GenBank (NR_111174, EU673110, and AY640258). Hence, both morphological and molecular characteristics confirmed the fungus as L. theobromae. To confirm fungal pathogenicity, ten 1-year-old healthy plants of C. oleifera were inoculated with the fungus. Mycelial plugs (5 mm) taken from a 7-day-old colony growing on PDA were deposited on wounds with a sterilized knife on leaves and covered with moist cotton. Ten additional control plants were treated similarly but with sterile PDA plugs. Plants were maintained in a moist chamber at 26 ± 2°C for 3 days and then in a greenhouse at 25°C and 40% relative humidity. All the inoculated plants produced typical leaf spot symptoms 3 weeks after inoculation. The fungus was consistently re-isolated from all inoculated plants. Control plants did not show any symptoms. L. theobromae has been reported to cause cankers and dieback in a wide range of hosts and is common in tropical and subtropical regions of the world (1,2), but not previously reported causing disease on C. oleifera. To our knowledge, this is the first report worldwide of leaf spot of C. oleifera caused by L. theobromae. References: (1) S. Mohali et al. For. Pathol. 35:385, 2005. (2) E. Punithalingam. Page 519 in: CMI Descriptions of Pathogenic Fungi and Bacteria. Commonwealth Mycological Institute, Kew, Surrey, UK, 1976.

First Report of Burkholderia andropogonis Causing Bacterial Leaf Spot of Betel Palm in Hainan Province, China.

In May 2009, a severe bacterial disease of arecanut (Areca catechu L.) with an incidence of 100% was observed in a plantation of about 8,400 plants in Wenchang City, Hainan Province, China (19°47.171' N, 110°54.335' E). Symptoms consisted of small circular to elongated brown lesions, ranging from 1 to 105 mm in length and 1 to 21 mm in width, surrounded by yellow halos. White colonies, without fluorescent or diffusible pigments, were consistently recovered on King's B Medium plates from lesions surface-sterilized in 70% ethyl alcohol for 1 min. All isolates were gram-negative and each had a single, polar, sheathed flagellum. Isolates were identified as a Burkholderia sp. based on physiological and biochemical tests: oxidase and catalase positive, negative for arginine dihydrolase, gelatin hydrolysis and starch hydrolysis, and negative for acid production from levan (1,3). Sequences (approx. 1,400 bp each) of the 16S rRNA gene amplified from four isolates using primer pair 27F/1492R (2) (GenBank Accession Nos. JX415481, JX415479, JX415482, and JX415483) shared 99% sequence identity with that of Burkholderia andropogonis strain 6369 (DQ786951). Representative isolates Y11 (China General Microbiological Culture Collection Center No. CGMCC 1.12337), Y30 (CGMCC 1.12338), W15, and W20 were compared with B. andropogonis strain NCPPB No. 1012 and all caused a hypersensitive reaction on leaves of Nicotiana benthamiana. Isolate pathogenicity was tested twice with a total of three replications per isolate. Two young leaves each of 2-year-old arecanut plants were infiltrated with a bacterial suspension of 108 CFU/ml, then covered individually with plastic bags for 48 h, and incubated at 100% relative humidity with 16 h of daylight at 25°C by day and 8 h of darkness at 20°C by night. After 7 days, small water-soaked spots with yellow halos were observed and 60 days after inoculation, lesions developed similar to those caused by B. andropogonis in the field. Koch's postulates were fulfilled by reisolating bacteria from typical lesions on inoculated plants. These bacteria were identical to inoculated strains in colony morphology and sequences of the 16S ribosomal RNA gene. To our knowledge, this is the first report of B. andropogonis infection on betel in Hainan Province, mainland China. This disease was first reported in Taiwan, a province of China. Conditions of high humidity and high temperature support disease outbreaks and infection can result in severe economic losses. In 2012, this disease also appeared on a number of plantations located in other counties. As betel is, economically, the second most important crop in Hainan Province, measures should be required to control this disease, especially in typhoon seasons. References: (1) S. H. Hseu et al. Plant Pathol. Bull. 16:131, 2007. (2) D. J. Lane. In: E. Stackebrandt, et al. Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Chichester, United Kingdom, pp. 115-175, 1991. (3) X. Li and S. H. De Boer. Plant Dis. 89:1132. 2005.

First Report of Stem Bleeding in Coconut Caused by Ceratocystis paradoxa in Hainan, China.

Stem bleeding of coconut was discovered in 2009 in Hainan, China. Affected trunk areas exhibited dark discoloration and a reddish brown or rust-colored liquid bleeding from different points. Stem tissues under the lesions rotted and became brownish yellow to black. Affected plants died within 3 to 4 months after stem symptoms first appeared. Stem bleeding of coconut is known to occur in production areas worldwide. The disease was first reported in Sri Lanka (1), caused severe damage to PB-121 hybrids in Indonesia (2), and is now known to occur in many other coconut-producing countries. However, to our knowledge, this is the first report of the disease in China. A fungus was isolated from lesion margins of diseased coconut trees. Colonies on potato dextrose agar (PDA) were white, became black 1 to 2 days later, and emitted a strong, fruity aroma. The fungus produced conidia, which were cylindrical, colorless to pale brown, and 6.9 to 14.9 × 3.1 to 6.0 µm, and oval, black chlamydospores that were 7.9 to 19.4 × 4.6 to 11.0 µm. The optimum temperature for mycelial growth ranged from 25 to 35°C and it did not grow at temperatures lower than 5°C or higher than 40°C. On the basis of these characteristics, the fungus was identified as Ceratocystis paradoxa (Dade) C. Moreau (anamorph Thielaviopsis paradoxa (de Seynes) Höhn). The internal transcribed spacer (ITS) region was amplified from genomic DNA with primers ITS1 and ITS4 and the PCR products were sequenced (GenBank Accession No. JQ039332). BLAST analysis showed 99% sequence similarity with C. paradoxa (GenBank Accession No. HQ248205.1). Pathogenicity of the fungus was tested by inoculating 10, 3-year-old coconut trees of the cv. green tall at the 12-leaf stage in the field. Agar plugs (5 mm in diameter) from the periphery of 7-day-old C. paradoxa colonies grown on PDA were placed on healthy trunks, rachis, and leaves, which were either wounded or unwounded. Wounds were made with a sterilized cork borer. Sites of the inoculations were wrapped with plastic tape to prevent desiccation; the experiment was repeated three times. Controls received plain PDA discs. Two weeks after inoculation, characteristic rusty brown lesions appeared only on wounded plants that were inoculated with the fungus. A brownish liquid oozed from the points of inoculation. Controls did not show signs of disease development. C. paradoxa was reisolated from the diseased tissues. Infection occurred on wounded sites only, suggesting that wounds may be required for infection. To prevent stem bleeding of coconut trees by C. paradoxa, vigilant cultural practices must be maintained to avoid causing wounds on the trees. References: (1) S. A. Alfieri. Plant Pathol. Circular No. 53. Florida Department of Agriculture Division of Plant Industry, 1967. (2) D. R. N. Warwick and E. E. M. Passos. Trop. Plant Pathol. 34:175, 2009.

[Analysis of sister-chromatid exchange rate from peripheral lymphocyte of people exposed to environmental cadmium].

Sister-chromatid exchanges (SCEs) were analyzed using peripheral lymphocyte from 38 persons (20 men and 18 women) who had been exposed to environmental cadmium (Cd), and 9 controls (7 men and 2 women). Correlations between SCE rates and urinary Cd-concentrations were done. There were no significant differences in SCE rates between the Cd-polluted and nonpolluted groups. There were no significant correlations between SCE rates and urinary Cd concentrations.

Cytogenetic investigation in lymphocytes of people living in cadmium-polluted areas.

Chromosome aberrations were analyzed from peripheral lymphocyte cultures of 21 men and 19 women who had been exposed to environmental cadmium, and 11 controls (9 men and 2 women). The average cadmium level in the urine of the Cd-polluted group was 3.32 micrograms/l for men and 3.83 micrograms/l for women. There were significant differences in chromosome aberration frequencies between the Cd-polluted and non-polluted groups. The number of individuals with relatively high aberration frequencies (greater than or equal to 5%) in the Cd-polluted group was greater than in the controls. Individuals with a high cadmium content in urine (greater than or equal to 3 micrograms/l) had higher aberration frequencies and more severe aberration types in comparison with the low-cadmium group (less than 3 micrograms/l). There were significant correlations between chromosome aberration frequencies and urinary cadmium content (r = 0.463). The linear regression equation was determined. Considering the conflicting results in other published reports, it is hard to say that the conclusion that cadmium only acts synergistically to enhance the mutagenicity of other compounds present in the environment is correct. According to our study, environmental cadmium cannot only induce chromosome aberrations but also increases the chromosomal aberration frequencies and the frequency of severe aberration types.