Vector population manipulation for control of arboviruses – a novel prospect for India

India, the seventh largest country in the world, has diverse geographical and climatic regions with vast rural and peri‐urban areas. Many are experiencing an escalation in the spread and intensity of numerous human diseases transmitted by insects. Classically, the management of these vector‐borne diseases is underpinned by either chemical insecticides and/or environmental management targeted at the vector. However, these methods or their present implementation do not offer acceptable levels of control, and more effective and sustainable options are now available. Genetic strategies for the prevention of arbovirus transmission are most advanced for dengue and chikungunya, targeting their primary vector, Aedes aegypti. The national burden in terms of morbidity and mortality as a direct consequence of dengue virus in India is considered to be the largest worldwide, over 4 times that of any other country. Presently, new genetic technologies are undergoing field evaluation of their biosafety and efficacy in several countries. This paper discusses the merits of these approaches and argues for fair and transparent appraisal in India as a matter of urgency. Identification of any associated risks and their appropriate mitigation are fundamental to that process. © 2013 Society of Chemical Industry     

Selective media for <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Selective media without pentachloronitrobenzene were developed for quantitative assays of Fusarium oxysporum in soils. Media Fo-G1 and Fo-G2 were effective for naturally infested soils, Fo-W1 and Fo-W2 for wild-type isolates in soils containing a nitrate-nonutilizing (nit) mutant, and Fo-N1 and Fo-N2 for nit mutants. Selective media were made using ammonium citrate dibasic, l-sorbose, econazole nitrate, 25% iminoctadine triacetate solution and 50% tolclofos-methyl wettable powder for soil dilutions of 100-fold or more (Fo-G1, FoW1 and Fo-N1) and 10-fold (Fo-G2, Fo-W2 and Fo-N2). Potassium chlorate was added to Fo-N1 and Fo-N2. The efficacy for selectively isolating F. oxysporum was confirmed using six soils naturally infested with one of six formae speciales of F. oxysporum and with soil dilutions containing conidia of wild-type strains or nit mutants from the six formae speciales. On Fo-G1 and Fo-G2, most colonies of F. oxysporum were compact and round with purplish or reddish pigment in the reverse. Cylindrocarpon sp. formed colonies as large as those of F. oxysporum but were distinguishable by their colony morphology. Other contaminants such as F. solani, F. moniliforme, and Trichoderma were suppressed by medium ingredients and colonies of F. oxysporum. On Fo-W1 and Fo-W2, colony morphology of F. oxysporum and contaminants corresponded to that on Fo-G1 and Fo-G2, although F. oxysporum failed to produce the pigment. On Fo-N1 and Fo-N2, nit mutants formed clear colonies from 100- and 10-fold soil dilutions, respectively, and contaminants seldom formed large colonies.     

Quorum sensing as a target for developing control strategies for the plant pathogen <Emphasis Type="Italic">Pectobacterium</Emphasis>

Quorum sensing is a regulatory mechanism that connects gene expression to cell density in bacteria. Amongst proteobacteria, numerous functions are regulated in this way, including pathogenicity in the Enterobacteriaceae genus Pectobacterium. In Pectobacterium, the signalling molecules involved in this regulatory process belong to the N-acyl-homoserine lactone class. Over the last 6 years, various studies have shown that these signal molecules could be degraded by other bacteria or by plant and animal cells, opening the path to innovative biocontrol strategies. This review explores the various determinants of pathogenicity in Pectobacterium and describes approaches that have been developed to quench the quorum-sensing-dependent pathogenicity in Pectobacterium. These approaches range from signal degradation by physicochemical constraints to the identification of signal-sensing inhibitors and from the identification of enzymes degrading acyl-homoserine lactones to the construction of transgenic plants tolerant to Pectobacterium.     

Coleoptile cuticle of barley is necessary for the increase in appressorial turgor pressure of <Emphasis Type="Italic">Blumeria graminis</Emphasis> for penetration

To determine whether the cuticle of the barley coleoptile is responsible for a rise in appressorial turgor pressure in Blumeria graminis, we determined the appressorial turgor pressure by measuring cytorrhysis and plasmolysis in the presence of PEG6000. Appressorial turgor pressure significantly increased 13–14 h after inoculation. On the other hand, when the cuticle was completely removed from the barley coleoptile surface with diethyl ether, turgor pressure did not increase. Moreover, when we then recoated the surface with the exogenous barley cuticle fraction, appressorial turgor pressure significantly increased 12–13 h after inoculation. These results suggest that the cuticle on the surface of the barley coleoptile is necessary for the increase in the appressorial turgor pressure.     

‘Universal’ spray droplet adhesion model – accounting for hairy leaves

The adhesion of a spray droplet upon initial contact with a leaf surface is extremely important to spray efficacy and is dependent on dynamic interactions between droplets (formulation, size, velocity) and leaf (micro‐topography, surface chemistry, veininess, hairiness and orientation). A ‘universal’ spray droplet adhesion model has previously been developed, using 50% aqueous acetone contact angles as a measure of leaf surface properties; this model satisfactorily predicts initial adhesion over a range of formulation surface tensions, droplet sizes and velocities. However, it failed to fit data from hairier leaves. This study investigates initial spray droplet adhesion on hairy leaves. Two categories of hairy leaves were identified by how the droplets penetrate the leaf hairs, Wenzel (hairy) and Cassie–Baxter (super hairy). For the Wenzel‐type, a simple constant accounted for the increased droplet shatter caused by the hairs. For the Cassie–Baxter‐type, a cushioning factor was introduced to account for the absorption of kinetic energy at impact by the hair mat. The cushioning factor was estimated by measuring the relative height of the hair mat. By including these two parameters, the new model successfully predicted the mean adhesion of non‐hairy, hairy and super‐hairy plants (R² = 0.96). This model and the underlying principles determining hairy leaf adhesion developed in this article will help develop spray formulations effective at targeting hairy‐leaved weed and crop species.     

Application of real‐time RT‐PCR for large‐scale testing of potato for Potato spindle tuber pospiviroid*

The real‐time RT‐PCR protocol of Boonham and coworkers performed extremely well in a recent ring test, comparing different methods for detection of Potato spindle tuber pospiviroid (PSTVd) in several laboratories. Since, in addition, real‐time PCR technology has proved suitable for high‐throughput testing, this method was chosen as the starting point for the development of a protocol for the large‐scale testing of potato. The initial experiments focused on the specificity of the primers and probes with regard to different isolates of PSTVd and other (pospi‐) viroids. Further experiments were performed with leaf material from both primarily and secondarily infected plants. The parameters studied were sampling position, growing‐on temperature and bulking rate. In addition, different grinding, nucleic‐acid extraction and disinfection methods were compared. To monitor false negatives and positives, different controls were included and tested in duplex and triplex formats. The final protocol was tested using a hundred samples from the Dutch potato‐monitoring programme. The results of this pilot experiment were promising. Future plans include the development of a protocol for direct tuber testing and inter‐laboratory ring testing of the protocols.     

Genetic analysis of an attenuated <Emphasis Type="Italic">Papaya ringspot virus</Emphasis> strain applied for cross-protection

Papaya ringspot virus (PRSV) HA 5-1, a nitrous acid-induced mild mutant of severe strain HA, widely applied for control of PRSV by cross-protection, was used to study the genetic basis of attenuation. Using infectious clones, a series of recombinants was generated between HA 5-1 and HA and their infectivity was analyzed on the systemic host papaya and the local lesion host Chenopodium quinoa. The recombinants that contained mutations in P1 and HC-Pro genes caused attenuated infection on papaya without conspicuous symptoms, similar to HA 5-1. The recombination and sequence analyses strongly implicated two amino acid changes in the C-terminal region of P1 and two in HC-Pro of HA 5-1 involved in the attenuated infection on papaya. The recombinants that infected C. quinoa plants without local lesions contained the same mutations in the C-terminal region of HC-Pro for attenuated infection on papaya. We conclude that both P1 and HC-Pro bear important pathogenicity determinants for the infection on the systemic host papaya and that the mutations in HC-Pro affecting pathogenicity on papaya are also responsible for the inability to induce hypersensitive reaction on C. quinoa.     

Characteristics for Practical Use of Attenuated Isolate UA-Fukushima of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis>

L₁₁A-Fukushima (L₁₁A-F) derived from attenuated isolate LuA of Tomato mosaic virus (ToMV) has the highest ability to cross protect against virulent ToMV among LuA and its derivatives and is stably inherited. Growth, yield, fruit quality and symptom attenuation of inoculated tomato plants did not differ significantly between L₁₁A-F and L₁₁A. The infectivity of progeny viruses in tomato infected with LuA-F was less than 4% of that with virulent ToMV. From these results, L₁₁A-F appears to possess the properties necessary for practical use. To manage L₁₁A-F strictly, a PCR-based assay to detect trace contamination of virulent ToMV in L₁₁A-F preparations was established. Received 10 June 2002/ Accepted in revised form 30 October 2002     

基于AHP-IE-MEA模型的矿山生态安全风险预警

基于对矿山生态安全风险因素的分析,找出了风险预警的关键环节,通过指标筛选构建了矿山生态安全风险预警指标体系并划分了各指标的五级预警区间,在此基础上设计了AHP-IE-MEA预警模型,最后把该模型应用在神东集团所属的三家煤矿,测算其生态安全风险预警等级。实证分析表明:M1矿处于四级预警,M2和M3矿处于无警情状态。研究表明,指标体系和AHP-IE-MEA模型适合于矿山生态安全风险预警,具有合理性和可行性,该方法的提出将为测算矿山生态安全风险等级提供参考。     

A diagnostic medium for the semi-selective isolation and enumeration of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis>pv. <Emphasis Type="Italic">vignicola</Emphasis>

A semi-selective medium for isolation of Xanthomonas axonopodis pv. vignicola from cowpea (Vigna unguiculata) plant and soil samples was developed. Twelve carbon and five nitrogen sources were tested with four strains of X. axonopodispv.vignicola, and 25 antibiotics were screened against saprophytes. -cellobiose (10g) was selected as the optimal carbon source. Among the antibiotics, cefazoline inhibited growth of most of the saprophytes with little effect on strains of the pathogen. ,-methionine enhanced growth of X. axonopodispv.vignicola. Boric acid along with ammonium chloride suppressed growth of Pseudomonas fluorescens. The semi-selective medium designated as cefazoline-cellobiose-methionine (CCM) medium contained K₂HPO₄ 1.34g, KH₂PO₄ 0.4g, MgSO₄ 0.3g, H₃BO₃ 0.2g, NH₄Cl 1.0g, -cellobiose 10g, cycloheximide 0.2g, ,-methionine 1.0g, cefazoline 10mg and agar 14g per l of water (pH 7.2). Colonies of X. axonopodispv.vignicola on CCM medium were whitish, round, raised and 0.2–1.8mm in diameter 96h after incubation. CCM medium generally inhibited growth of Pantoea agglomerans, Bacillus subtilis and saprophytes isolated from cowpea leaves. Colonies of Pseudomonas fluorescens and a saprophytic bacterium, which were not completely suppressed by CCM, could be differentiated from X. axonopodispv.vignicola by their smaller size and different color. The CCM medium proved useful for isolation of X. axonopodispv.vignicola from cowpea plant and soil samples. This is the first report of a semi-selective medium developed for detection of X. axonopodispv.vignicola.     

Desmodium species and associated biochemical traits for controlling Striga species: present and future prospects

We discovered serendipitously a new and highly effective intervention against Striga spp., including Striga hermonthica (African witchweed), in cereals, which involves intercropping with cattle fodder legumes, Desmodium spp., including D. uncinatum . Although soil shading and additional nitrogen made some contribution to the reduction of S. hermonthica infestation, an allelopathic mechanism associated with the intercrop was a major factor. Root exudates of D. uncinatum contain novel flavonoid compounds, some of which stimulate germination of S. hermonthica and others dramatically inhibit its subsequent development, including radicle growth. Desmodium spp. have been developed as intercrops for both maize and sorghum and are now being evaluated for millet. From the experience with Desmodium spp., there is now the possibility for producing edible legumes suitable for intercropping with maize and other cereals to respond to a broader profile of farmer practices. These legumes would incorporate the powerful S. hermonthica controlling properties of D. uncinatum through feasible breeding programmes, with appropriate contributions from analytical chemistry to plant molecular genetics and biotechnology. In the longer term, it may also be possible to transfer genes associated with the allelopathic attributes to cereals themselves by heterologous gene expression, creating a new generation of parasitic weed-free cereals.     

Calcium/calmodulin-dependent signaling for prepenetration development in <Emphasis Type="Italic">Cochliobolus miyabeanus</Emphasis> infecting rice

Cochliobolus miyabeanus forms a specialized infection structure, an appressorium, to infect rice. Contacting a hard surface induces appressorium formation in C. miyabeanus, while the hydrophobicity of the substratum does not affect this morphogenic infection event. To determine whether the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. miyabeanus, the effects of a calcium chelator (ethylene glycol tetraacetic acid; EGTA), phospholipase C inhibitor (neomycin), intracellular calcium channel blocker (TMB-8), calmodulin antagonists (chlorpromazine, phenoxybenzamine, and W-7), and calcineurin inhibitor (cyclosporin A) on morphogenesis and infection were examined. Addition of Ca²⁺ and the calcium ionophore A23187 did not affect conidial germination, while the number of appressoria decreased with higher concentrations. EGTA inhibited conidial germination and appressorium formation. The calcium channel blocker did not affect appressorium formation at any concentration; however, calmodulin antagonists and the calcineurin inhibitor specifically reduced appressorium formation at the micromolar level. One of the calmodulin antagonists, W-7, also inhibited accumulation of mRNA of the calmodulin gene within germinating conidia and/or appressorium-forming germ tubes. Thus, biochemical processes controlled by the calcium/calmodulin signaling system seem to be involved in the induction of prepenetration morphogenesis on rice.     

Use of sclerotia of<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> for efficient production of conidia of<Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in liquid culture

A study was conducted to determine the feasibility of using sclerotia ofSclerotinia sclerotiorum for producing conidia ofConiothyrium minitans in liquid culture. The medium (SST) was made of water containing 2.0, 1.5, 1.0 or 0.5% (w/v) ground sclerotia ofS. sclerotiorum and 100 μgl ⁻¹ thiamine hydrochloride (HCl). One ml of conidial suspension (2 × 10⁷ conidia ml⁻¹) ofC. minitans LRC 2534 was inoculated into 100 ml of SST medium or control (thiamine HCl in water) and incubated at 20 ± 2°C on a shaker at 200 rpm. Subsamples were removed periodically and examined under a compound microscope. Conidia in the SST media germinated within 24 h, developed into branched hyphae within 48 h, produced pycnidia after 3–4 days, and the pycnidia released mature conidia after 7 days. Production of conidia varied with the concentration of sclerotia in the SST medium. It was lower (3.6 × 10⁶ conidia ml⁻¹) at 0.5% but higher (1.2 × 10⁸ conidia ml⁻¹) at 2%. The new conidia were viable and the colonies developing from them showed the original morphological characteristics. It was concluded that using SST liquid medium as a substrate for mass production of conidia ofC. minitans has potential for use in commercial development of this mycoparasite as a biocontrol product. http:www.phytoparasitica.org posting Jan. 23, 2007.     

<Emphasis Type="Italic">Ulocladium atrum</Emphasis> as a biological control agent for white mold of bean caused by<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Field studies were conducted near Lethbridge, Alberta, Canada, in 2001, 2004 and 2005 to determine the efficacy of the antagonistic fungusUlocladium atrum for control of white mold of bean caused bySclerotinia sclerotiorum. Results of the 3 years of field trials showed that, compared with the untreated control, foliar application of a spore suspension ofU. atrum (300 ml m⁻² of 10⁶ spores ml⁻¹ suspension) significantly reduced incidence and severity of white mold, increased seed yield and reduced contamination of bean seed by sclerotia ofS. sclerotiorum. The level of control of white mold observed in the treatment ofU. atrum was similar to that of the mycoparasitic fungusConiothyrium minitans, but lower than the fungicide treatments of Ronilan (vinclozolin) at the rate of 1200 g ha⁻¹ per application in 2001, or Lance (boscalid) at the rate of 750 g ha⁻¹ per application in 2004 and 2005. The potential for use ofU. atrum as a biological control agent for sclerotinia diseases is discussed. http://www.phytoparasitica.org posting Nov. 12, 2006.     

Chlorpyrifos-methyl plus bioresmethrin; Methacrifos; Pirimiphos-methyl plus bioresmethrin; and synergised bioresmethrin as grain protectants for wheat

Field trials with various pesticide combinations were carried out on bulk wheat in commercial silos in Queensland, South Australia and Western Australia. Laboratory bioassays on samples of treated grain at intervals over 8 months using malathion-susceptible and malathion-resistant strains established the following orders of efficacy: against Sitophilus oryzae (L.), chlorpyrifos-methyl 10 mg kg^?1 + bioresmethrin 1 mg kg^?1 = methacrifos 15 mg kg^?1 in aerated storage > pirimiphos-methyl 4 or 6 mg kg^?1 + bioresmethrin 1 mg kg^?1 = bioresmethrin 4 mg kg^?1 + piperonyl butoxide 16 mg kg^?1; against Rhyzopertha dominica (F.), bioresmethrin 4 mg kg^?1 + piperonyl butoxide 16 mg kg^?1 > methacrifos 15 mg kg^?1 > chlorpyrifos-methyl 10 mg kg^?1 + bioresmethrin 1 mg kg^?1 = pirimiphos-methyl 4 or 6 mg kg^?1 + bioresmethrin 1 mg kg^?1. All treatments completely prevented production of progeny in Sitophilus granarius (L.), Tribolium castaneum (Herbst), T. confusum Jackquelin du Val and Oryzaephilus surinamensis (L.). The biological efficacy of methacrifos was greater and the rate of degradation lower in aerated than in non-aerated storage. Residue levels of all compounds were determined chemically and were below proposed international residue levels to be considered by the Codex Alimentarius Commission.     

<Emphasis Type="Italic">In vitro</Emphasis> screening for sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis>L.) resistant calli to <Emphasis Type="Italic">Diaporthe helianthi</Emphasis> fungal culture filtrate

Diaporthe helianthi the causal agent of sunflower (Helianthus annuus) stem canker, causes significant reductions in yield and oil content in most sunflower-growing areas. With the aim of enhancing host resistance, we selected in vitro sunflower calli against culture filtrates of two pathogen isolates (7/96 and 101/96). This technique may be an effective and rapid tool to discriminate the most virulent D. helianthi isolate and to screen for host resistance in the early stage of a breeding programme. Further investigation on the mechanisms involved in defence pathways showed no induction of salicylic acid and pathogenesis-related proteins in calli, indicating that the host resistance is not associated with Systemic Acquired Resistance but probably other biochemical mechanisms.     

Plants for planting; indirect evidence for the movement of a serious forest pathogen, <Emphasis Type="Italic">Teratosphaeria destructans</Emphasis>, in Asia

Fungal diseases caused by native pathogens and pathogens introduced with planting stock have a significant impact on exotic plantation forestry in the tropics. Teratosphaeria destructans (formerly Kirramyces destructans) is a serious pathogen causing leaf, bud and shoot blight diseases of Eucalyptus spp. in plantations in the sub-tropics and tropics of south-east Asia. This pathogen was first discovered in Indonesia in 1995 and has subsequently spread to Thailand, China, Vietnam and East Timor. The biology, ecology and genetics of this important pathogen have not been explored yet. The objective of this study was, thus, to determine the genetic diversity and movement of T. destructans throughout south-east Asia using multi-gene phylogenies and microsatellite markers. Out of nine gene regions only two microsatellite markers detected a very low nucleotide polymorphism between isolates; seven other gene regions, ITS, β-tubulin, EF1-α, CHS, ATP6 and two microsatellite loci, reflected genetic uniformity. The two polymorphic molecular markers resolved six haplotypes among isolates from Indonesia and only a single haplotype elsewhere in Asia. The low diversity observed among isolates in the region of the first outbreak is as expected for a small founder population. The spread of a single clone over large distances throughout the region supports the hypothesis of spread via the human-mediated movement of germplasm.