Recombinase Polymerase Amplification assay for rapid detection of a geminivirus associated with potato apical leaf curl disease

Journal of Plant Diseases and Protection - Recombinase Polymerase Amplification (RPA) assay was developed for specific detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV [potato]),...     

Theaflavins depolymerize microtubule network through tubulin binding and cause apoptosis of cervical carcinoma HeLa cells

Here we studied the antiproliferative activity of theaflavins in cervical carcinoma HeLa cells by investigating their effects on cellular microtubules and purified goat brain tubulin. Theaflavins inhibited proliferation of HeLa cells with IC(50) value of 110 ± 2.1 μg/mL (p = < 0.01), caused cell cycle arrest at G(2)/M phase and induced apoptosis with alteration of expression of pro- and antiapoptotic proteins. Along with these antiproliferative activities, theaflavins act as microtubule depolymerizers. Theaflavins disrupted the microtubule network accompanied by alteration of cellular morphology and also decreased the polymeric tubulin mass of the cells. The polymerization of cold treated depolymerized microtubules in HeLa cells was prevented in the presence of theaflavins. In vitro polymerization of purified tubulin into microtubules was also inhibited by theaflavins with an IC(50) value of 78 ± 2.43 μg/mL (P < 0.01). Thus, disruption of cellular microtubule network of HeLa cells through microtubule depolymerization may be one of the possible mechanisms of antiproliferative activity of theaflavins.     

Honey Bee (Hymenoptera: Apidae) Nursing Responses to Cuticular Cues Emanating from Short-term Changes in Larval Rearing Environment

Honey bee larvae are dependent on the social structure of colony for their provisioning and survival. With thousands of larvae being managed collectively by groups of foragers (collecting food resources) and nurse bees (processing food and provisioning larvae), coordination of colony efforts in rearing brood depends on multiple dynamic cues of larval presence and needs. Much of these cues appear to be chemical, with larvae producing multiple pheromones, major being brood ester pheromone (BEP; nonvolatile blend of fatty acid esters) that elicits both short-term releaser effects and long-term primer effects. While BEP can affect colony food collection and processing with the signaling of larval presence, it is unclear if BEP signals individual larval needs. To understand this aspect, in a series of experiments we manipulated larval feeding environment by depriving larvae from adult bee contact for 4-h period and examined (1) nurse bee interactions with contact-deprived and nondeprived larvae and larval extracts; (2) forager bee responses to contact-deprived and nondeprived larval extracts. We also characterized BEP of contact-deprived and nondeprived larvae. We found that nurse honey bees tend to aggregate more over contact-deprived larvae when compared with nondeprived larvae, but that these effects were not found in response to whole hexane extracts. Our analytical results suggest that BEP components changed in both quantity and quality over short period of contact deprivation. These changes affected foraging behavior, but did not appear to directly affect nursing behavior, suggesting that different chemical cues are involved in regulating nursing effort to individual larvae.     

Molecular markers for late blight resistance breeding of potato: an update

Late blight is the most devastating disease of the potato crop that can be effectively managed by growing resistant cultivars. Introgression of resistance (R) genes/quantitative trait loci (QTLs) from the Solanum germplasm into common potato is one of the plausible approaches to breed resistant cultivars. Although the conventional method of breeding will continue to play a primary role in potato improvement, molecular marker technology is becoming one of its integral components. To achieve rapid success, from the past to recent years, several R genes/QTLs that originated from wild/cultivated Solanum species were mapped on the potato genome and a few genes were cloned using molecular approaches. As a result, molecular markers closely linked to resistance genes or QTLs offer a quicker potato breeding option through marker‐assisted selection (MAS). However, limited progress has been achieved so far through MAS in potato breeding. In near future, new resistance genes/QTLs are expected to be discovered from wild Solanum gene pools and linked molecular markers would be available for MAS. This article presents an update on the development of molecular markers linked to late blight resistance genes or QTLs by utilization of Solanum species for MAS in potato.     

A process for controlling intracellular bacterial infections induced by membrane injury

Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and replication of many pathogens. We found that the lysosomal synaptotagmin Syt VII is required for a mechanism that promotes phagolysosomal fusion and limits the intracellular growth of pathogenic bacteria. Syt VII was required for a form of Ca2+-dependent phagolysosome fusion that is analogous to Ca2+-regulated exocytosis of lysosomes, which can be triggered by membrane injury. Bacterial type III secretion systems, which permeabilize membranes and cause Ca2+ influx in mammalian cells, promote lysosomal exocytosis and inhibit intracellular survival in Syt VII +/+ but not -/- cells. Thus, the lysosomal repair response can also protect cells against pathogens that trigger membrane permeabilization.     

Disease progression of a lethal decline caused by the 16SrIV‐D phytoplasma in Florida palms

Recently, a new phytoplasma was discovered in Hillsborough County in the state of Florida, USA. This phytoplasma belongs to the 16SrIV taxonomic group and is classified as subgroup D. It is the causal agent of lethal bronzing disease (LBD) of palm. Since the discovery of LBD in 2006, the disease has spread throughout much of the state. In 2014 and 2015, stands of cabbage palm and queen palms that had been present at the University of Florida's Fort Lauderdale Research and Education Center in Davie, FL began showing symptoms of LBD. After confirming the presence of the LBD phytoplasma in initially infected palms by nested PCR and RFLP analysis, all palms were systematically sampled over the period of 1 year to monitor and quantify disease spread. A total of 30 cabbage palms were tested monthly by qPCR, with five testing positive on the first sample date. By the end of the study period, 16 cabbage palms had died from the infection. A total of 16 queen palms were surveyed, with three palms initially testing positive. By the end of the study, four queen palms had tested positive and died from the infection. To the authors’ knowledge, this study is the first to document and quantify spread of palm‐infecting phytoplasmas. This data provides important insights into the ecology of palm‐infecting phytoplasmas and highlights the impact that the movement of infective insects can pose to established stands of palms.     

Development of simplified and rapid nucleic acid release protocol for PCR based detection of <Emphasis Type="Italic">Potato viruses</Emphasis>

A simple, cost-effective and rapid viral nucleic acid release (NAR) buffer suitable for RT-PCR based diagnostic assay was developed for the detection of potato viruses. The NAR buffer and commercially available RNA isolation kit were compared for RT-PCR based assay, where an amplicon of expected size (~380 bp) targeting PVY was observed in both isolations indicating that it can be used in RT-PCR based diagnostic assays. The same was further validated for its repeatability by running across more than hundred suspected potato leaf samples collected from different sources where, it showed consistent results for the presence of PVY indicating its reliability. The NAR buffer assay was examined for its sensitivity in comparison with the kit based isolation where both the assays were able to detect even up to 10^?5 dilution without affecting the sensitivity. NAR buffer was found stable up to 28 days at -20 °C and for 14 days at 4 °C without losing PCR sensitivity. The assay was also found effective to release the nucleic acid from potato leaves, thrips and aphids for PCR and RT-PCR based detection of DNA viruses like ToLCNDV-potato and other RNA viruses. The developed protocol is simple, less laborious, time-saving (10-15 min) and economical (1/100th of kit) as compared to kit based protocol. The assay can be adopted in diagnostic laboratories for detection of RNA/DNA viruses from potato plants and in thrips as well.     

Potato bacterial wilt in India caused by strains of phylotype I, II and IV of Ralstonia solanacearum

Bacterial wilt or brown rot is one of the most devastating diseases of potato caused by a bacterium Ralstonia solanacearum (Smith 1986) Yabuuchi et al. (Microbiol Immunol 39:897–904 1995). Traditionally, R. solanacearum is classified into five races (r) on the basis of differences in host range and six biovars (bvs) on the basis of biochemical properties. Recently using molecular methods, R.?solanacearum has been classified into phylotypes based on the intergenic transcribed sequence of the ribosomal RNA genes 16S and 23S and into sequevars based on the endoglucanase gene (egl) sequence. In the present study, 75 bacterial strains, isolated from wilt infected potatoes from various potato growing regions of India, were classified by traditional and molecular methods. The identity of all the strains was confirmed as R. solanacearum as expected single 280-bp fragment resulted in all the strains following PCR amplification using R. solanacearum specific universal primer pair 759/760. Biovar (bv) analysis, based on utilization of disaccharide sugars and hexose alcohols, categorised the 75 strains into bv2 (78.7 %), 2 T (5.3 %), 3 (5.3 %) and 4 (10.7 %). The phylotype specific multiplex PCR assigned 78.7 % strains to phylotype II, 16.0 % to phylotype I and 5.3 % to phylotype IV. Phylogenetic analysis of egl gene sequences clustered all fifty nine phylotype II (bv2) strains with reference strain IPO1609 (IIB-1), all four phylotype IV (bv2T) strains with reference strain MAFF301558 (IV-8), three phylotype I (bv3) strains with reference strain MAFF211479 (I-30) and all eight phylotype I (bv4) and one phylotype I (bv3) strain with reference strain CIP365 (I-45). The study concluded that the Indian potato strains of R. solanacearum belong to three out of four phylotypes namely: the Asian phylotype I, the American phylotype II, and the Indonesian phylotype IV. This is the first study to address the diversity of R. solanacearum from potato in India using phylotype and sequevar scheme. We also report here for the first time the occurrence of phylotype IV sequevar 8 (bv2T) strain of R. solanacearum causing potato bacterial wilt in mid hills of Meghalaya in India.     

Tubifex production using agro-industrial wastes and raw cattle dung

ABSTRACT

Tubifex (Tubifex tubifex) was cultured in captivity using three different wastes: rice mill sludge (RMS), dairy sludge (DS), and raw cattle dung (RCD). Three experiments were conducted: 10, 20, and 30 days. A total of 100 g of tubifex at 62.5 g m^?2 was inoculated in 1.6 m² fiberglass-reinforced plastic tanks. Comparing all production parameters, RMS > DS > RCD. Growth rate (g m^?2 d^?1) did not differ among durations. RMS- and DS-fed tubifex contained higher protein and fat than RCD-fed tubifex. Efficiency on production of g tubifex per kg of waste material was highest at 10 days, declining with time for all waste materials. This experiment suggests that RMS and DS are effective wastes for tubifex culture, with total production increasing with no reduction in growth rate through 30 days, but with efficiency declining after 10 days.