Morphological and molecular characterisation of Pythium species causing chilli (Capsicum annuum L.) damping-off

Twenty-five Pythium isolates comprising five species viz., Pythium aphanidermatum, P. deliense, P. graminicola, P. heterothallicum and P. ultimum from different geographical locations of Tamil Nadu (Coimbatore, 4; Cuddalore, 6; Dindigul, 1; Dharmapuri, 1; Erode, 1; Madurai, 1; Namakkal, 7; Thanjavur, 1; Theni, 1; Thirunelveli, 1 and Vellore, 1) isolated from chilli crop were analysed with randomly amplified polymorphic DNA (RAPD) markers. Morphological and molecular characteristics of these different species were correlated with the RAPD. Polymerase chain reaction amplification of total genomic DNA with six random primers generated unique banding patterns depending on the primer and the isolate. The isolate I₁₇ produced identical banding patterns, while other isolates produced dissimilar bands within the particular species, indicating the genetic diversity among the isolates within a species. Morphological characters were also different from each other even in isolate I₁₇ which shared identical bands. Cluster analysis showed minimum and maximum per cent similarities among the tested Pythium species which ranged from 49 to 89%, respectively. RAPD markers were better suited for differentiating isolates within a species rather than species.     

Delimiting cryptic pathogen species causing apple Valsa canker with multilocus data

Fungal diseases are posing tremendous threats to global economy and food safety. Among them, Valsa canker, caused by fungi of Valsa and their Cytospora anamorphs, has been a serious threat to fruit and forest trees and is one of the most destructive diseases of apple in East Asia, particularly. Accurate and robust delimitation of pathogen species is not only essential for the development of effective disease control programs, but also will advance our understanding of the emergence of plant diseases. However, species delimitation is especially difficult in Valsa because of the high variability of morphological traits and in many cases the lack of the teleomorph. In this study, we delimitated species boundary for pathogens causing apple Valsa canker with a multifaceted approach. Based on three independent loci, the internal transcribed spacer (ITS), β‐tubulin (Btu), and translation elongation factor‐1 alpha (EF1α), we inferred gene trees with both maximum likelihood and Bayesian methods, estimated species tree with Bayesian multispecies coalescent approaches, and validated species tree with Bayesian species delimitation. Through divergence time estimation and ancestral host reconstruction, we tested the possible underlying mechanisms for fungal speciation and host‐range change. Our results proved that two varieties of the former morphological species V. mali represented two distinct species, V. mali and V. pyri, which diverged about 5 million years ago, much later than the divergence of their preferred hosts, excluding a scenario of fungi–host co‐speciation. The marked different thermal preferences and contrasting pathogenicity in cross‐inoculation suggest ecological divergences between the two species. Apple was the most likely ancestral host for both V. mali and V. pyri. Host‐range expansion led to the occurrence of V. pyri on both pear and apple. Our results also represent an example in which ITS data might underestimate species diversity.     

Identification and host range of causal agent of dieback disease on Jatropha curcas

Diseased Jatropha branches collected from Institute for Agricultural Research (IAR), Samaru, Zaria field were plated in Potato Dextrose Agar with Streptomycin (PDAS) and incubated for seven days. Grown cultures were observed under microscope and with aid of identification manuals Fusarium sp. was isolated. Inoculated seedlings showed symptoms of dieback. The fungus was also inoculated on some crop plants and it induced the typical dieback symptoms on sesame and leaf spot/ blight on groundnut and soya beans, respectively.     

Morphological and molecular characterisation of Lasiodiplodia theobromae from various banana cultivars causing crown rot disease in fruits

Twelve isolates of Lasiodiplodia theobromae were collected from 12 different commercial banana cultivars belonging to various banana growing regions of Tamil Nadu, India. The results revealed that most of the L. theobromae isolates exhibited significant differences in morphology, colour and spore size. However, all 12 isolates took 3–4 days invariably to cover the 90 mm Petri plates. Among the 12 isolates, the isolates LT3a from Robusta and LT10c from dwarf Cavendish produced abundant pycnidia. These isolates were also analysed through Random amplified polymorphic DNA (RAPD) markers for genetic diversity. Polymerase chain reaction amplification of total genomic DNA of the 12 isolates of L. theobromae with 14 random primers generated numerous polymorphisms among the isolates, while many of the intense bands were shared among majority of the isolates. Cluster analysis also indicated a high degree of genetic variability within L. theobromae isolates from different banana cultivars. The 12 isolates were separated into three clusters namely A, B and C. The maximum similarity index of 80% was recorded between the isolates LT7d from Karpuravalli and LT10c from dwarf Cavendish. The least similarity index of 29.41% was recorded with the isolates LT1f isolated from Poovan and LT5a isolated from Virupakshi. Within the species of L. theobromae the genetic variability was high and it underlines and validates existence of significant intra-specific diversity in isolates of L. theobromae infecting different banana cultivars.     

Production,purification and biochemical characterisation of a novel lipase from a newly identified lipolytic bacterium Staphylococcus caprae NCU S6

A novel lipase, SCNL, was isolated from Staphylococcus caprae NCU S6 strain in the study. The lipase was purified to homogeneity with a yield of 6.13% and specific activity of 502.76 U/mg, and its molecular weight was determined to be approximately 87 kDa. SCNL maintained above 80% of its initial activity at a wide range of temperatures (20–50 °C) and pH values (6–11), with an optimal temperature at 40 °C and optimal pH at 9.0 with p-nitrophenyl palmitate as a substrate. SCNL exhibited a higher residual activity than the other staphylococcal lipases in the presence of common enzyme inhibitors and commercial detergents. The lipase activity was enhanced by organic solvents (isooctane, glycerol, DMSO and methanol) and metal ions (Na⁺, Ba²⁺, Ca²⁺, and Mn²⁺). The Km and Vmax values of SCNL were 0.695 mM and 262.66 s⁻¹mM⁻¹, respectively. The enzyme showed a preference for p-NP stearate, tributyrin and canola oil. These biochemical features of SCNL suggested that it may be an excellent novel lipase candidate for industrial and biotechnological applications.     

Biochemical, physiological and morphological variation in unarmed hymenolepids (Eucestoda: Cyclophyllidae)

The genus Hymenolepis contains a number of unarmed species. These frequently possess similar morphologies and are difficult to discriminate using the traditional method of comparative morphology. A parasite of the long-tailed field mouse, Apodemus sylvaticus in northeast Ireland, resembles the widespread H. diminuta which is usually a parasite of the rat. Analysis of general and specific proteins of the adults in A. sylvaticus , laboratory mice and rats suggests that the parasite found in the former host and H. diminuta are genetically distinct, though more closely allied than either is to H. nana, H. citelli and H. microstoma . Experimental analysis of the growth and expulsion of the Irish material and H. diminuta from SPF C57 laboratory mice, rats and wild caught A. sylvaticus suggests that there are behavioural and physiological differences in these taxa. Both are expelled from C57 mice though the hymenolepid from Irish A. sylvaticus persists for 3 days more than those of H. diminuta . The former prospers better in rats than H. diminuta in A. sylvaticus . Detailed comparison of the gross morphology of cysticercoid and adult H. diminuta and the Irish hymenolepid reveals differences in size rather than qualitative attributes. The occurrence of H. diminuta in A. sylvaticus is discussed. It is concluded that the hymenolepid recovered from Irish A. sylvaticus differs sufficiently from H. diminuta to warrant species status and that it has adapted to the alimentary canal of A. sylvaticus . This cestode material is described under the name of H. hibernia sp. nov.     

Molecular characterisation of Trichoderma species using SRAP markers

Trichoderma are commonly used as bio control agents in various agro ecosystems. They are known to produce a variety of compounds that induce resistance responses in plants. Among different species of Trichoderma, T. harzianum, T. viride, T. koningii and T. hamatum are commercially used as bio control agents. In the present study, four commercially important species of Trichoderma isolated from coffee ecosystem were screened with sequence related amplified polymorphism (SRAP) markers. Among 48 SRAP primer pairs tested, 29 primers were polymorphic and generated 316 distinct scorable fragments. Out of 347 amplified fragments, 177 fragments were found polymorphic with an average of 6.10 fragments per primer combination. The average polymorphism information content (PIC) and resolving power (Rp) of the 29 polymorphic SRAP primer pair were 0.42 and 14.62, respectively. The UPGMA dendrogram clearly divided Trichoderma species into two broad clusters. The highest homology (83.0%) was observed between T. viride and T. Harzianum and the lowest homology (74.0%) was observed between T. Harzianum and T. konangii. Further, among 29 polymorphic SRAP markers screened, four primer pairs (ME1-EM3, ME1-EM20, ME1-EM22 and ME2-EM4) produced unique fragments specific to each species. These markers can be useful in easy and rapid identification of the species.     

Molecular mechanisms of interspecies transmission and pathogenicity of influenza viruses: Lessons from the 2009 pandemic

The emergence of the 2009 H1N1 virus pandemic was unexpected, since it had been predicted that the next pandemic would be caused by subtype H5N1. We also had to learn that a pandemic does not necessarily require the introduction of a new virus subtype into the human population, but that it may result from antigenic shift within the same subtype. The new variant was derived from human and animal viruses by genetic reassortment in the pig, supporting the concept that this animal is the mixing vessel for the generation of new human influenza viruses. Although it is generally believed that the 2009 outbreak was mild, there have been severe cases particularly among the young and the middle-aged. Pathogenicity and host range are determined to a large extent by the polymerase, the haemagglutinin and the NS1 protein of influenza A viruses. There is evidence that mutations of these proteins may change the pathogenicity of the new virus.     

Molecular characterisation and disease severity of leptospirosis in Sri Lanka

Leptospirosis is a re-emerging zoonotic disease all over the world, important intropical and subtropical areas. A majority of leptospirosis infected patients presentas subclinical or mild disease while 5-10% may develop severe infection requiringhospitalisation and critical care. It is possible that several factors, such as theinfecting serovar, level of leptospiraemia, host genetic factors and host immuneresponse, may be important in predisposition towards severe disease. DifferentLeptospira strains circulate in different geographical regionscontributing to variable disease severity. Therefore, it is important to investigatethe circulating strains at geographical locations during each outbreak forepidemiological studies and to support the clinical management of the patients. Inthis study immunochromatography, microscopic agglutination test and polymerase chainreaction were used to diagnose leptospirosis. Further restriction fragment lengthpolymorphism and DNA sequencing methods were used to identify the circulating strainsin two selected geographical regions of Sri Lanka. Leptospira interrogans,Leptospira borgpetersenii and Leptospira kirschneristrains were identified to be circulating in western and southern provinces.L. interrogans was the predominant species circulating in westernand southern provinces in 2013 and its presence was mainly associated with renalfailure.     

Erwinia amylovora strains from outbreaks of fire blight in Spain: phenotypic characteristics

One hundred and thirty strains of Erwinia amylovora recovered from Spanish foci of fire blight from 1995 to 2000 were characterised and compared to reference strains from different sources and origins. Their rapid identification was performed by double antibody sandwich indirect (DASI) ELISA, using specific monoclonal antibodies against E. amylovora, and molecular confirmation by PCR using primers specific to the native plasmid pEA29. The Spanish strains of E. amylovora grew on different general and selective media producing typical colonies, except one of them that was deficient in levan production, whereas none of them grew on minimal agar medium with copper sulphate and low content of asparagine. All of them were susceptible to tetracycline, streptomycin, kasugamycin and oxolinic acid. Biochemical characterisation of selected strains by API 20E system revealed a great homogeneity, with 80% of the Spanish strains showing one of the two majority API 20E profiles described for E. amylovora, and the remaining strains showing minor differences. Pathogenicity on pear fruits and hypersensitivity reaction was confirmed, but a delayed reaction was observed for two Spanish strains. This is the first characterisation of a large collection of Spanish strains of E. amylovora.     

限气贮藏猕猴桃果实伤害产生机制的研究

采用ＰＥ袋方法于常温一藏秦猕猴桃,贮藏期间袋内ＣＯ２浓度高于６％时果实会产生伤害贮藏前期,果实的果肉和果心硬度均很高,这与两部位淀粉酶和多聚半乳糖醛酸酶活性严重被抑制淀粉和果胶物质水解被抑制有关。贮藏中期,果肉出现水浸状,到贮藏末期,果心和照相机人均很快腐烂,这主要与Ｖｃ含量明显降低,导致膜脂过氧化加剧有关。而与ＳＯＤ活性变化关系不大。而贮藏期间,果心硬度明显高于果肉,这与果心淀粉酶和多聚半乳醛酸     

Rapid preliminary characterisation of host specificity of leaf-beetles (Coleoptera: Chrysomelidae)

Rapid characterisation of host specificity is important both in biological control of weeds and studies in ecology and evolution. A means for doing this was developed and tested on four species of leaf beetles of interest for biological control of the weed Mimosa pigra (Mimosaceae). We identified the most promising for the more detailed tests necessary to obtain release approval. The impact of time-dependent effects and effects of experience were also investigated as part of this study but were not detected. Short-term host specificity tests on adult feeding accurately predicted the results of longer term trials. The long-term trial showed that survival on a plant species depended on feeding on it. Hence short-term feeding trials can predict the longer term survival of adults on other plant species. Different feeding results were obtained in cut foliage versus entire plants but no consistent pattern was shown: of the two insect species tested, one species ate more of the cut material while the opposite was demonstrated for the second species. Species in order of priority for further consideration as biocontrol agents were Syphrea bibiana, Genaphthona sp., Syphrea sp. and Paria sp. The latter three species were probably not sufficiently specific for release. The tests done here allow the identification of the most likely species upon which to conduct the more laborious and difficult larval developmental tests, saving considerable resources.     

Biochemical and physiological studies of soluble esterases fromDrosophila melanogaster

Twenty-two soluble esterases have been identified inD. melanogaster by combining the techniques of native polyacrylamide gel electrophoresis and isoelectric focusing. The sensitivity of each isozyme to three types of inhibitors (organophosphates, eserine sulfate, and sulfydryl reagents) identified 10 as carboxylesterases, 6 as cholinesterases, and 3 as acetylesterases. Three isozymes could not be classified and no arylesterases were identified. The carboxyl- and cholinesterases could each be further divided into two subclasses on the basis of inhibition by organophosphates and sulfhydryl reagents, respectively. Cholineand acetylesterases have characteristic substrate preferences but both subclasses of carboxylesterases are heterogeneous in substrate utilization. Subclass 2 carboxylesterases exhibit diverse temporal expression patterns, with subclass 1 carboxylesterases generally found in larvae and subclass 1 cholinesterases and acetylesterases more characteristic of pupae and adults. Tissues showing the greatest number of isozymes are larval body wall (eight) and digestive tract (six in larvae, six in adults). Carboxylesterases are distributed across a wide range of tissues, but subclass 1 cholinesterases are generally associated with neural or neurosecretory tissues and subclass 2 cholinesterases with digestive tissues.This study was funded in part by the Rural Credits Development Fund.     

Simple and efficient genomic DNA extraction protocol for molecular characterisation of Phytophthora colocasiae causing taro leaf blight

Genomic DNA extraction protocol with relatively high quantity and purity is prerequisite for the successful molecular identification and characterisation of plant pathogens. Conventional DNA extraction methods are often time-consuming and yield only very poor quantity of genomic DNA for samples with higher mycelial age. In our laboratory, we have aimed at establishing an efficient DNA isolation procedure, exclusively for the oomycete pathogen Phytophthora colocasiae causing serious leaf blight disease in taro. For this a phenol free protocol was adopted, which involves SDS/Proteinase K-based inactivation of protein contaminants, extraction of nucleic acids using chloroform: isoamyl alcohol and later precipitation of genomic DNA using isopropanol and sodium acetate. The purity of the isolated DNA was analysed by A_260/280 and A_260/230 spectrophotometric readings and confirmed by restriction digestion with restriction enzyme Eco RI. In this study, a comparative assessment was done with CTAB method and the commercial genomic DNA purification kit (Thermo Fisher Scientific, Fermentas, EU). The extracted DNA was found to be suitable for further downstream applications like ITS amplification of the rDNA ITS region and PCR amplification with species-specific primers.     

A novel milky disease organism from Australian scarabaeids: Field occurrence,isolation, and infectivity

A novel milky disease organism has been found causing disease in Aphodius tasmaniae and other scarabaeid larvae in the field in Australia. The sporangium is exceptionally long, measuring 10.5 × 1.5 μm, with a small central spore, measuring 1.0 × 0.6 μm. The vegetative cell is about half the size of the sporangium. The disease was easily transmitted by injection of spores into the hemocoel, with typically milky symptoms developing in 2–4 weeks. Spores will form in vivo at temperatures down to 12°C. For A. tasmaniae third-instar larvae, the ID₅₀ by injection was 3 × 10² spores/larva, yet no infection resulted when larvae were reared in peat containing up to 10⁸ spores/g, i.e., the disease was not successfully transmitted per os. All 10 species of scarabaeids tested were susceptible to the disease when spores were injected; however, all attempts to infect larvae per os were unsuccessful. In vitro culture was also unsuccessful.     

Pythium and Phytopythium species associated with weeds collected in vegetable production fields in Brazil

This study aimed to identify Pythium and Phytopythium species from weeds collected in vegetable fields and test their pathogenicity. Weeds with symptoms of damping-off, root rot or wilt were sampled in the Brazilian states of Ceará, Goiás and Pernambuco, as well as in the Distrito Federal, for isolation and identification of the causal agents. Once isolated, colonies with typical Pythium and Phytopythium characteristics grew in selective V8 medium. Procedures for species identification included morphology and amplification of the ITS and Cox II regions, which were compared with other accessions available at GenBank. The phylogenetic relationships among the isolates and pathogenicity to their original hosts were evaluated. Six Pythium species were identified: P. aphanidermatum, P. oopapillum, P. orthogonon, P. ultimum var. ultimum, P. myriotylum and P. sylvaticum, and two species of Phytopythium, Phy. chamaehyphon and Phy. oedochilum. In the pathogenicity tests, the 10 weed hosts showed symptoms of damping-off or root rot after inoculation, with exception of Portulaca oleraceae in which none of the isolates was pathogenic. Therefore, common weeds in vegetable fields areas can host different Pythium and Phytopythium species and play an important role in the epidemiology of vegetable diseases, in particular on pathogen survival and population increase.