Molecular Characterization and Recombination Analysis of the Complete Genome of Apple Chlorotic Leaf Spot Virus

The complete nucleotide sequence of an Indian isolate of Apple chlorotic leaf spot virus (ACLSV) was determined and found to be 7,525 nt in length. The genome organization was similar to known isolates of ACLSV, encoding three ORFs. Comparisons indicated high sequence variability among known isolates with overall nucleotide sequence identities of 80 to 84%. A striking variable region was identified among the replicase protein upstream of the RNA‐dependent RNA polymerase (aa 1510–1590), which showed a 41–43% match with the corresponding region in other isolates. Phylogenetic analysis at the nucleotide level clustered the isolates into three groups, without any relation to geographical origin. Recombination analysis showed that the isolate is a recombinant with recombination sites spread throughout the genome, especially in the polymerase gene region (nt 4700–5400). Most recombination sites were bordered by an upstream region (5′) of GC‐rich and downstream region (3′) of AU‐rich sequences of similar length. Correlation of recombination site with host type is discussed, and it was found that there were more interlineage recombinations in the apple host compared with intralineage recombinations.     

Occurrence and Diversity of Pome Fruit Viruses in Apple and Pear Orchards in Latvia

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus are economically important viruses infecting fruit tree species worldwide. To evaluate the occurrence of these pome fruit viruses in Latvia, a large‐scale survey was carried out in 2007. Collected samples were tested for infection by DAS ELISA and multiplex RT‐PCR. The accuracy of the detection of the viruses in multiplex RT‐PCR was confirmed by sequencing amplified PCR fragments. The results showed a wide occurrence of viruses in apple and pear commercial orchards established from non‐tested planting material. More than 89% of the tested apple trees and more than 60% of pear trees were infected with one or more pome fruit viruses. Analyses showed that the high occurrence of viruses in several apple cultivars is due to the propagation of infected clonal rootstocks and scions from infected mother trees. Sequence analyses targeting the 3′‐terminal region of the tested viruses showed various degrees of genetic diversity within respective virus isolates. This is the first report of the occurrence of ACLSV, ASGV and ASPV in apple and pear trees in Latvia and demonstrates their genetic diversity in different host genotypes.     

Metschnikowia bicuspidata and Enterococcus faecium co-infection in the giant freshwater prawn Macrobrachium rosenbergii

In May 2001, an epizootic yeast and bacterial co-infection in the giant freshwater prawn Macrobrachium rosenbergii occurred in Taiwan causing a cumulative mortality of 25%. The diseased prawns had a yellowish-brown body color, milky hemolymph, opaque, whitish muscles, and were approximately 7 mo old with total lengths ranging from 8 to 10 cm. Histopathological examination showed marked edema, yeast infiltration, and necrotic lesions with inflammation in the muscles, hepatopancreas and other internal organs. We isolated 2 pathogens from the diseased prawns, one was a yeast (AOD081MB) and the other a gram-positive coccus (AOD081EF). The gram-positive coccus was identified as Enterococcus faecium by the API 20 Strepsystem, conventional biochemical tests, and it had 99% 16S rDNA sequence identity (GenBank Accession Number AJ276355) to E. faecium (GenBank Accession Number AF529204). The sequence of a PCR product from the D1/D2 domain of 26S rDNA (GenBank Accession Number AF529297) from the yeast gave 99% sequence identity to Metschnikowia bicuspidata (GenBank Accession Number U44822). Experimental infections with these isolates produced gross signs and histopathological changes similar to those observed in the naturally infected prawns. The lethal doses (LD50) for isolate E. faecium AOD081EF, M. bicuspidata AOD081MB and the co-infection were 4.7 x 10(4), 2.6 x 10(2), and 2.4 x 10(2) colony-forming units prawn(-1), respectively. This is the first report of a confirmed co-infection of M. bicuspidata and E. faecium in prawn aquaculture.     

Molecular variability analyses of <Emphasis Type="Italic">Apple chlorotic leaf spot virus</Emphasis> capsid protein

The complete sequences of the coat protein (CP) gene of 26 isolates of Apple chlorotic leaf spot virus (ACLSV) from India were determined. The isolates were obtained from various pome (apple, pear and quince) and stone (plum, peach, apricot, almond and wild Himalayan cherry) fruit trees. Other previously characterized ACLSV isolates and Trichoviruses were used for comparative analysis. Indian ACLSV isolates among themselves and with isolates from elsewhere in the world shared 91–100% and 70–98% sequence identities at the amino acid and nucleotide levels, respectively. The highest degree of variability was observed in the middle portion with 9 amino acid substitutions in contrast to the N-terminal and C-terminal ends, which were maximally conserved with only 4 amino acid substitutions. In phylogenetic analysis no reasonable correlation between host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other Trichoviruses revealed the TaTao ACLSV peach isolate to be phylogenetically closest to Peach mosaic virus, Apricot pseudo chlorotic leaf spot virus and Cherry mottle leaf virus. Recombination analysis (RDP3 ver.2.6) done for all the available ACLSV complete CP sequences of the world and Indian isolates indicate no significant evidence of recombination. However, one recombination event among Indian ACLSV-CP isolates was detected. To the best of our knowledge, this is the first report of complete CP sequence variability study from India and also the first evidence of homologous recombination in ACLSV.     

Identification and Molecular Characterization of a New Geminivirus Betasatellite Infecting Malvastrum coromandelianum in China

The complete nucleotide sequence of a satellite molecule associated with Malvastrum leaf curl Guangdong virus (MLCuGdV) infecting M. coromandelianum plants exhibiting leaf curl symptoms in a suburb of Guangzhou, Guangdong Province of China, is described and analysed. The molecule has typical features of betasatellites, containing a single ORF in the complementary‐sense strand, an A‐rich region, the satellite‐conserved region and a stem–loop structure. Compared with the geminivirus betasatellites in GenBank database, this molecule shows the highest nucleotide sequence identity of 71.9% with Tomato leaf curl Philippine betasatellite isolate Laguna1 (ToLCPB, AB307732). Phylogenetic analysis indicates that it is more related to isolate Laguna 1 and Laguna 2 of ToLCPB. According to the proposed species demarcation threshold of betasatellites (78% nucleotide identity), it is a novel betasatellite species, for which we propose the name Malvastrum leaf curl Guangdong betasatellite (MLCuGdB).     

Biological Characterization and Complete Genome Sequence of a Possible Strain of Indian cassava mosaic virus from Jatropha curcas in India

The outbreak of a severe mosaic disease with a significant incidence was noticed on Jatropha curcas plants growing in Lucknow, Northern India. The causal virus was successfully transmitted by whiteflies (Bemisia tabaci) and grafting from naturally infected to healthy J. curcas plants. The association of Begomovirus with the mosaic disease of J. curcas was detected by PCR using primers specific to DNA‐A of Begomoviruses. Further, full‐length DNA‐A genome of ～2.7 kb was amplified by RCA followed by digestion with Bam HI restriction enzyme. Cloning and sequencing of obtained amplicons resulted in 2740 nucleotides of complete DNA‐A consisting of six ORFs and IR region (GenBank Accession HM230683 ). The sequence analysis revealed highest 85% similarities with Jatropha curcas mosaic virus, 77–84% with Indian cassava mosaic virus and 73–76% with Sri Lankan cassava mosaic virus isolates. Phylogenetic analysis of the Begomovirus isolate also showed a clear‐cut distinct relationship with earlier reported Begomoviruses from Jatropha curcas and other Begomoviruses. On the basis of the guidelines of the International Committee on Taxonomy of Viruses (ICTV‐2008), our virus isolate was identified as a possible strain of Indian cassava mosaic virus, and its name Jatropha mosaic India virus (JMIV) is proposed.     

Detection and Molecular Variability of Apple Stem Grooving Virus in Shaanxi,China

Apple stem grooving virus (ASGV) is one of the economically important latent viruses that are distributed in apple production areas worldwide. The presence of ASGV in apple trees was studied by serological assay and molecular biology methods. A total of 550 apple leaf samples from 14 different areas in Shaanxi were tested by DAS‐ELISA, and the results revealed an ASGV infection level of 55%. Those samples were also examined by RT‐PCR, and an infection level of 67% was found. Fourteen complete coat protein gene sequences of ASGV were obtained; phylogenetic analysis revealed that these 14 sequences separated into two clusters regardless of the geographic origin or host plants. To our knowledge, this is the first report of molecular variability analysis of ASGV in apple trees in China.     

Evidence of Cotton leaf curl Burewala virus Variant and its Associate Betasatellite Causing Yellow Mosaic of Eggplant (Solanum melongena) in Pakistan

Eggplant (Solanum melongena L.) plants with severe leaf mosaic and mottling were found in a kitchen garden near cotton fields in Pakistan. Rolling Circle Amplification products from six of the naturally infected eggplant plants, subjected to PCR, successfully amplified expected products of 2.8 and 1.4 kb using begomovirus and betasatellite‐specific primers, respectively. Based on 99% nucleotide sequence identity, the virus was identified as a variant of Cotton leaf curl Burewala virus (CLCuBuV) (GenBank Accession No. HG428709). Likewise, the sequenced betasatellite with a maximum of 97% nucleotide sequence identity was recognized as a new variant of Cotton leaf curl Multan betasatellite (CLCuMuB^Mul) (GenBank Accession No. HG428708). The symptomatic induction of Cotton leaf curl disease in CLCuBuV susceptible cotton genotype CIM‐496 by back‐indexing further confirmed the presence of CLCuBuV in eggplant. This is the first report of CLCuBuV and its associate betasatellite in naturally infected plants of eggplant.     

Evaluation of the Use of Ammonium Bicarbonate and Oregano (Origanum vulgare ssp. hirtum) Extract on the Control of Apple Scab

In vitro experiments showed that ammonium bicarbonate and aqueous extracts of oregano were effective in inhibiting conidia germination and germ‐tube elongation of Venturia inaequalis. Complete inhibition was achieved by 1% ammonium bicarbonate, 2% oregano extract and 0.01% synthetic fungicide difenoconazole. Two orchard experiments were conducted on the highly susceptible cv. Mutsu to apple scab to investigate the efficacy of ammonium bicarbonate alone or in combination with an aqueous extract of oregano for the control of apple scab. In 2008 and 2009, except for the applications of 1% aqueous extract of oregano, the applications of ammonium bicarbonate (0.5 and 1%) and difenoconazole (0.01%) to trees at 10‐day intervals significantly reduced disease incidence and severity on leaves and fruit compared to the water‐treated control. In both years, the efficacy of 0.5 and 1% ammonium bicarbonate in inhibiting both disease incidence and severity on leaves and fruit was equally effective in all monthly assessments from June to September. Combining 0.5 and 1% ammonium bicarbonate with 1% aqueous extract of oregano did not significantly improve the efficacy of stand‐alone applications of treatments in the final assessment in 2008 and 2009. All treatments were neither phytotoxic to leaves and fruit nor did they adversely affect quality parameters of fruit including physiological disorders and taste both at harvest and after storage. These results indicate that ammonium bicarbonate treatment may be applied as an alternative chemical for the control of apple scab.     

Efficacy of Boric Acid,Monopotassium Phosphate and Sodium Metabisulfite on the Control of Apple Scab

In vitro experiments indicated that boric acid, monopotassium phosphate, sodium metabisulfite and synthetic fungicide fluopyram + tebuconazole were effective in inhibiting conidia germination and germ‐tube elongation of Venturia inaequalis. Monopotassium phosphate even at the highest concentration used in the study reduced conidia germination and germ‐tube elongation of V. inaequalis by 22.1% and 28.8%, respectively; however, the difference between two compounds at lower concentrations except 0.05% (for conidia germination) and 0.1% (for germ‐tube elongation) of boric acid was statistically significant (P ≤ 0.05). Complete inhibition was achieved by 0.01% sodium metabisulfite, 0.035% fluopyram + tebuconazole and 0.2% boric acid. Two orchard trials were conducted on the highly susceptible cv. Mutsu to apple scab to ascertain the efficacy of 0.2% boric acid, 0.5% monopotassium phosphate, 0.5% sodium metabisulfite and 0.035% fluopyram + tebuconazole for the control of apple scab. In both 2013 and 2014, except for the applications of monopotassium phosphate and sodium metabisulfite, the applications of boric acid and fluopyram + tebuconazole to trees at 10‐day intervals significantly reduced disease incidence and severity on leaves and fruit compared to the water‐treated control. In both years, the efficacy of boric acid and fluopyram + tebuconazole treatments was similar in reducing both disease incidence and severity on leaves and fruit in all monthly assessments from July to September. All treatments were neither phytotoxic to leaves and fruit nor did they adversely affect quality parameters of harvested fruit. These results show that boric acid treatment may be applied as an alternative chemical for the control of apple scab.     

Biological and Molecular Characterization of Alfalfa Mosaic Virus Infecting Trumpet Creeper (Campsis radicans) in Iran

Samples of trumpet creeper (Campsis radicans) leaves showing mottling and mosaic were collected from plants growing in a private garden in Tehran province, Iran, in 2012. Symptomatic leaf samples were tested for Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV) and Peanut stunt virus (PSV) infection in enzyme‐linked immunosorbent assay (ELISA), using specific antibodies. None of the samples were positive for CMV and PSV; however, all reacted positively with that of AMV antiserum. In biological assay, systemic infection was found on Datura stramonium, Nicotiana tabacum cvs., White Burley, and Xanthi, 21 days postinoculation (DPI), while necrotic local lesions were obtained following inoculation of Phaseolus vulgaris and Vigna unguiculata within three to four DPI. Using a pair of primers specific for AMV, a DNA fragment of 880 bp was RT‐PCR‐amplified. Analysis of the sequences revealed the presence of 657 nucleotides of AMV complete coat protein (CP) gene (translating 218 amino acid residues). Phylogenetic analysis using neighbour‐joining (NJ) method clustered AMV isolates into two main types and the IRN‐Tru (GenBank Accession No. JX865593 ) isolate fell into type I. Pairwise nucleotide distances also confirmed two main types with the highest and lowest similarities for type I and II, respectively. The association of AMV with mosaic disease of C. radicans represents the first record from the world.     

Complete Genome Sequence of a Novel Wild tomato mosaic virus Isolate Infecting Nicotiana tabacum in China

Virus particles of approximately 740–760 nm in length and 13 nm in diameter were observed from a diseased Nicotiana tabacum (tobacco) plant in Sichuan Province, China. The complete genomic sequence of the virus isolate XC1 was determined to contain 9659 nucleotides without 3′ terminal poly(A) tail. XC1 has a genome typical of members of the genus Potyvirus, encoding a large polyprotein of 3075 amino acids. Putative proteolytic cleavage sites and a number of well characterized functional motifs were identified by sequence comparisons with those of known potyviruses. Sequence comparison revealed that XC1 shared the highest level of nucleotide sequence identity (76.5%) with Wild tomato mosaic virus (WTMV). Phylogenetic analysis showed that XC1 was closely related to the WTMV Guangdong isolate with an identity of 94.3% between CP gene sequence of the two viruses. We thus named XC1 WTMV‐XC‐1 as a novel isolate of WTMV. The full sequence of WTMV‐XC‐1 may serve as a basis for future investigations on the gene diversity of WTMV.     

Transmission of the Phytoplasma Associated with Bunchy Top Symptom of Papaya by Empoasca papayae Oman

Transmission tests were conducted with field‐collected Bunchy Top Symptoms (BTS) phytoplasma‐infected specimens of Empoasca papayae. BTS developed in all eight inoculated papayas 3 months later. The BTS phytoplasma was identified in six of eight inoculated papayas, whose partial 16S rRNA sequence (GenBank Accession no. FJ6492000 ) was 99.9% identical with those from the collected papayas (GenBank Accession no FJ649198 ) and E. papayae (GenBank Accession no. FJ649199 ), all of which are members of group 16SrII, ‘Candidatus Phytoplasma aurantifolia’. Results confirmed the ability of E. papayae to transmit the BTS phytoplasma.     

The distribution of Athetis lepigone and prediction of its potential distribution based on GARP and MaxEnt

Athetis lepigone (Möschler) is a new agronomic pest which has caused serious damages to summer maize in China. In order to effectively monitor it, it is necessary to carry‐out a worldwide investigation on its potential geographical distribution. In this study, we give two ecological niche models, Genetic Algorithm for Rule‐set Production and Maximum Entropy (MaxEnt), to predict the potential geographical distribution of A. lepigone. The results indicate that the suitable areas for A. lepigone are mainly in China (Beijing, Tianjin, central and southern Hebei, northern Jiangsu, Shandong, most of Henan, northern Anhui, central and southern Shanxi, central and southern Shaanxi, small parts of north‐west in Hubei, eastern Gansu, parts of Ningxia, western of Xinjiang and Dandong, and Liaoning) and other Asian countries (South Korea, North Korea and Japan). Parts of Europe (south‐western Russia, Ukraine, Moldova, Romania, Hungary, Bulgaria, Slovenia, and Croatia, Bosnia and Herzegovina, Serbia, parts of Austria, western Poland, the Czech Republic, Germany, northern Italy, Denmark, western Lithuania, Latvia, Estonia, southern Finland and Sweden) are also highly suitable for A. lepigone. In addition, in the states of the USA including Michigan, New York, Chicago, Missouri, Ohio, Illinois and Indiana are also favourable for its occurrence although till now no reports about of this species has been recorded. A jackknife test in MaxEnt showed that the mean temperature of the driest quarter was the most important environmental variable affecting the distribution of this pest.     

Identification of Colletotrichum Species Causing Bitter Rot of Apple and Pear in Croatia

Symptoms of bitter rot were observed on apple and pear fruits in the field and in storage in Croatia between 2009 and 2011. Fifteen Colletotrichum isolates from apple and two from pear were collected and identified by sequencing of the ITS1 and ITS2 regions of the ribosomal DNA. Phylogenetic analysis revealed that ten isolates from apple and two isolates from pear could be identified as Colletotrichum fioriniae, five isolates from apple were clustered in Colletotrichum clavatum, while one isolate was in the Colletotrichum acutatum A7 group. All isolates caused typical bitter rot symptoms when inoculated on apple and pear fruits.     

Molecular Analysis of ORF6, ORF7 and ORF8 of Beet yellows virus Iranian Isolate

Beet yellows virus (BYV), a member of the Closteroviridae family, is one of the most important sugar beet yellowing viruses. The nine ORFs of BYV genome encode different proteins required for BYV life cycle. We sequenced a part of the genome of BYV Iranian isolate consisting of ORF6, ORF7 and ORF8. The primer pair BYVA/Z was used for amplification of this region in RT‐PCR. The amplicon (1615 bp) was cloned and sequenced. Comparisons showed the amplified segment is corresponding to ORF6, ORF7 and ORF8 of BYV genome encoding coat protein, p20 and p21 proteins, respectively. The ORF7 of BYV Iranian isolate overlaps with ORF6 and ORF8 in four and 26 nucleotides at 5′ and 3′ ends, respectively. The ORF7 of Iranian isolate of BYV was sequenced completely. However, approximately 24 nt. from the beginning of ORF6 and 23 nt. from end of ORF8, including the stop codon, were not determined. ORF6, ORF7 and ORF8 showed the highest similarity at nucleotide (98.3, 99.4 and 99.2%) and amino acid (97.4, 98.9 and 100%) sequence levels, with BYV Ukrainian isolate. Phylogenetic analysis of the deduced amino acid sequences of ORF6, ORF7 and ORF8 revealed closer relationship of Iranian isolate of BYV with BYV Ukrainian isolate than other BYV isolates available at GenBank.     

昆明地区两栖动物多样性及保护研究

昆明地区共有21种两栖动物,隶2目8科12属。区系特点是：物种多样性很丰富,多于我国9个省区;东洋界种类有20种占绝对优势,没有古北界种类,古北东洋两界广布种仅1种;明显具西南区的特色有16种,特有种多,呈贡嵘螈及滇螈是昆明特有种,也是云南特有种,另有滇蛙等12种为中国特有种;模式产地为昆明的种类多,有7种;姬蛙科属种多,共3属4种。该文阐述了昆明为模式产地的种类现状,还提出了昆明地区两栖类的保护对策。