Fruit infestation of Acacia tortilis (Forsk) hyne by Bruchidius andrewesi Pic. (Coleoptera: Bruchidae) in the Thar desert

Bruchidius andrewesi Pic. has been recorded as a serious pest of pods and seeds of Acacia tortilis in the Thar desert of India. Pest infestation on developing pods and its relationship with morphological traits is reported. Pod infestation varied from 5 to 19% with 5–29% infestation of seeds. Infestation of pods is directly related to infestation of seed (r = 0.72^**), and both pod and seed infestation are also directly correlated with loss in seed biomass (r = 0.79^** and R = 0.88^**). The infestation of pods starts in November and increases steady until harvest. Seeds kept in the laboratory for further studies were found to be 100% infested with B. andrewesi, as the insect multiplied faster under these conditions. The heavy infestation is damaging not only to A. tortilis but also to other leguminous trees of the desert. Bruchidius andrewesi has also been found on pods and seeds of Prosopis cineraria, an important indigenous tree of the region.     

Diofenolan: a novel insect growth regulator in common citrus butterfly, <Emphasis Type="Italic">Papilio demoleus</Emphasis>

Penultimate and last instar larvae of the common citrus swallowtail butterfly, Papilio demoleus L. (Papilionidae: Lepidoptera), were treated with different doses of a novel juvenoid, diofenolan. Several deformities were observed as a result of topical administration of diofenolan which include delay in larval–larval and larval–pupal ecdysis, ecdysial failure, mortality, severe reduction in pupation, deformed pupae and complete inhibition of adult emergence. The juvenoid diofenolan severely hampers the normal growth, development and metamorphosis of P. demoleus and can be incorporated into integrated pest management programs for successful control of this important pest of citrus all over the world.     

OsSCE1 Encoding SUMO E2-Conjugating Enzyme Involves in Drought Stress Response of Oryza sativa

Small ubiquitin-like modifier (SUMO)-conjugating enzymes are involved in post-translational regulatory processes in eukaryotes, including the conjugation of SUMO peptides to protein substrate (SUMOylation). SUMOylation plays an important role in improving plant tolerance to abiotic stress such as salt, drought, heat and cold. Herein, we reported the isolation of OsSCE1 (LOC_Os10g39120) gene encoding a SUMO-conjugating enzyme from rice (Oryza sativa cv. Nipponbare) and its functional validation in response to drought stress. The E2 enzyme, OsSCE1, is one of three key enzymes involved in the conjugation of SUMO to its target proteins. Activated SUMO is transferred to the cysteine of an E2 enzyme and then to the target lysine residue of the substrate, with or without the help of an E3 SUMO ligase. Expression of OsSCE1 was strongly induced by polyethylene glycol 6000 (PEG6000) treatment, which suggested OsSCE1 may be involved in the drought stress response. Overexpression of OsSCE1 (OsSCE1-OX) in Nipponbare reduced the tolerance to drought stress. Conversely, the drought tolerance was slightly improved by the knockdown of OsSCE1 (OsSCE1-KD). These results were further supported by measurement of proline content in OsSCE1-OX and OsSCE1-KD transgenic lines under induced drought stress, which showed OsSCE1-KD transgenic lines accumulated higher proline content than the wild type, whereas OsSCE1-OX line had lower proline content than the wild type. These findings suggested OsSCE1 may play a role as a negative regulator in response to drought stress in rice.     

Experimental evaluation of the culture performance of three exotic and a ‘local’ Giant Freshwater Prawn (Macrobrachium rosenbergii,De Man 1879) culture strain in Fiji

Giant freshwater prawn (GFP; Macrobrachium rosenbergii) aquaculture has expanded rapidly since 1990. Most local culture industries, however, have developed in an unsystematic way. Fiji has a small culture industry producing the ‘Anuenue’ strain; however, performance of this strain has never been systematically evaluated. Recently, some Fijian farmers have reported declines in stock productivity. The current project evaluated the relative performance of three exotic strains with different genetic backgrounds from Malaysia, Indonesia and Vietnam, against the ‘local’ strain in Fiji in a 4 × 3 replicated pond trial experiment. A total of 5827 prawns were harvested after 143 days growout. Individual growth rate and relative survival of the Fiji strain were not statistically different from any of the introduced strains, but Vietnam strain was superior to that of the Malaysia strain. Genetic diversity showed significant differences in variability among strains, with the Malaysian strain displaying the lowest genetic diversity. Indonesia strain showed that females were reaching maturation earlier than other strains and were smaller in size. This study suggests that Malaysian and Indonesian strains would constitute a poor choice for Fiji, whereas the Vietnam strain consistently performed well on all criteria measured. High variation among replicate ponds within strains unfortunately confounded among‐strain variation.     

Development of conventional and real-time PCR protocols for specific and sensitive detection of Colletotrichum capsici in chilli (Capsicum annuum L.)

Anthracnose, caused by Colletotrichum capsici, is a major disease of chilli (Capsicum annuum L.) affecting both fruit and seed quality. The pathogen is both internally and externally seedborne. However, a rapid and sensitive method for detection of this pathogen in seeds is currently limited. In this study, a polymerase chain reaction (PCR) method based on sequence characterized amplified region (SCAR) marker was developed for specific and sensitive detection of C. capsici in chilli seeds and fruits. The developed SCAR primers were highly specific to C. capsici and resulted in the amplification of an expected 250-bp fragment from genomic DNA of all seven of the C. capsici isolates tested. No amplification occurred when the SCAR primers were tested with genomic DNA from three other fungal isolates and four other Colletotrichum species. The SCAR primers successfully amplified similar sized fragments from DNA derived from C. capsici-infected chilli fruits. The molecular detection sensitivity of C. capsici was 1 pg of purified C. capsici DNA template and 25 ng of DNA from C. capsici-infected chilli fruits. A real-time PCR assay was also developed using SYBR Green chemistry for detection of C. capsici in chilli fruits and seeds. The standard curve obtained showed a linear correlation between copy number of the cloned target DNA sequence of C. capsici and cycle threshold (Ct) values, with R² of 0.98. These PCR-based assays may be highly useful in detection of this important pathogen in chilli seeds and fruits in plant quarantine laboratories.     

The extent of genetic diversity among Vanilla species: Comparative results for RAPD and ISSR

Vanilla is a large genus of about 110 species in the orchid family (Orchidaceae), including the species Vanilla planifolia from which commercial vanilla flavoring is derived. Since most species of vanilla are considered rare and endangered there is an urgent need to conserve them through genetic analysis and propagation/conservation studies on this crop.The present study investigated the genetic diversity among nine leafy- and leaf-less Vanilla species employing 30 decamer RAPD primers and 10 ISSR primers. The species under study were diverse and displayed a range of variability (0–66% and 0–81% for RAPD and ISSR, respectively). A total of 154 RAPD polymorphic markers (83.24%, h = 0.378) and 93 ISSR polymorphic markers (86.11%, h = 0.363) were used to generate a genetic similarity matrix followed by the cluster analysis. Specific groupings were revealed by each cluster analysis with slight variation between two different markers. Among the nine species studied, V. planifolia, Vanilla aphylla and Vanilla tahitensis revealed very low level of variation within their collections, thus indicating a narrow genetic base. The large genetic distance of Vanilla andamanica from other species suggests its different origin. A close genetic affinity was observed between the pairs V. planifolia, V. tahitensis and Vanilla albida, V. aphylla. These are the first comparative results for RAPD and ISSR reporting inter-relationship among nine cultivated, wild and hybrid Vanilla species.     

Molecular phylogeny in Indian Citrus L. (Rutaceae) inferred through PCR-RFLP and trnL-trnF sequence data of chloroplast DNA

The taxonomy and phylogeny of Indian Citrus is revisited using PCR-RFLP of the trnD-trnT and rbcL-ORF 106 regions as well as sequence data analysis of the trnL-trnF intergenic spacer region of cpDNA. The study was based on 50 accessions of Citrus genotypes, collected from wild, semi-wild and domesticated stocks. Of the 13 restriction enzymes (RE) used for restriction digestion of the polymerase chain reaction (PCR) amplicons, four (Hinf I, Msp I, Alu I, Hae III) generated 47 restriction fragments, of which 24 (51%) were polymorphic. PCR-RFLP data showed a genetic distance ranging from 0 to 0.79 among 50 accessions of Citrus, and a cluster analysis, based on Neighbor-Joining (NJ) method, placed all the accessions in eight major clusters. Analysis of trnL-trnF sequences from 23 representative accessions of Citrus showed a pair-wise sequence divergence rate in the range of 0–0.064. NJ, minimum evolution (ME) and maximum parsimony (MP) analyses of trnL-trnF sequences produced phylogenetic trees, which placed all the 23 accessions in five clusters. PCR-RFLP analysis resulted in a well resolved phylogenetic tree with branches supported by moderate to high bootstrap values, while the trnL-trnF sequence-based trees showed only moderate to low bootstrap support for the internal tree branches, indicating uncertain origin of some Citrus genotypes. This study shows that the trnL-trnF spacer sequence data can detect genetic variation in Indian Citrus genotypes, but the utility of the data in inferring phylogeny at intra and inter-specific levels is limited probably by factors such as hybridization, bud mutations, apomixis and polyploidy. However, PCR-RFLP and trnL-trnF data supported the recognition of C. maxima, C. medica, and C. reticulata as the basal species of edible Citrus.     

Development and evaluation of an indirect enzyme-linked immunosorbent assay for detection of foot-and-mouth disease virus nonstructural protein antibody using a chemically synthesized 2B peptide as antigen.

Forty peptides were synthesized corresponding to hydrophilic clusters of amino acids within the sequences of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP). Six peptides were studied in more detail and the most promising, a 2B peptide, was evaluated in enzyme-linked immunosorbent assay (ELISA) using sera from naive, vaccinated, and vaccinated-and-challenged cattle as well as bovine sera from field outbreaks. The performance of the new NSP peptide ELISA was compared to that of 4 commercial NSP ELISA kits. Antibody to 2B was detectable from the end of the first week to the second week after infection in most of the nonvaccinated animals and by the second to third week in vaccinated-and-challenged animals. The sensitivity of the 2B peptide ELISA was comparable to the 3ABC Ceditest (Ceditest FMDV-NS, Cedi Diagnostics B.V.; Chung et al., 2002). With some modification and further validation, this 2B test could be useful as a screening or conformational NSP test in postvaccination surveillance for FMD.     

Construction and characterization of a fowlpox virus field isolate whose genome lacks reticuloendotheliosis provirus nucleotide sequences

Fowlpox virus (FWPV) has been isolated from vaccinated chicken flocks during subsequent fowlpox outbreaks that were characterized by a high degree of mortality and significant economic losses. This inability of current vaccines to induce adequate immunity in poultry could be reflective of an antigenic and/or biologic distinctiveness of FWPV field isolates. In this regard, whereas an infectious reticuloendotheliosis virus (REV) provirus is present in the majority of the field viruses' genomes, only remnants of REV long terminal repeats (LTR) have been retained in the DNAs of each vaccine strain. Although it has not been demonstrated whether the partial LTRs can provide an avenue for FWPV to reacquire the REV provirus by homologous recombination, utilizing viruses of which genomes lack any known integrated retroviral sequences could resolve concern over this issue. Therefore, such an entity was created by genetically modifying a recently isolated field strain of FWPV. This selection, in lieu of a commercial vaccine virus, as the progenitor was based on the probability that a virus circulating in the environment would be more antigenically similar to others in this locale and thus might be a better candidate for vaccine development. A comparison in vivo of the pathogenic traits of the parental wild-type field isolate, its genetically modified progeny, and a rescue mutant in whose genome the REV provirus was inserted at its previous location, indicated that elimination of the provirus sequence correlated with reduced virulence. However, even with elimination of the parasitic REV, the modified FWPV was still slightly more invasive than a commercial vaccine virus. Interestingly, both types of attenuated FWPV elicited a similar degree of antibody production in inoculated chickens and afforded them protection against a subsequent challenge by a field virus, the origin of which was temporally and geographically distinct from that of the progenitor strain. Due to its antigenicity being retained despite a decrease in virulence, this REV-less FWPV could potentially be developed as a vaccine against fowlpox.