Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.     

Improved Chiral SFC Screening for Analytical Method Development

In this study we describe the evaluation of a recently developed supercritical fluid chromatography (SFC) instrument for automated chiral SFC method development. The greatly improved gradient dwell volume and liquid flow control of the new instrument in combination with the use of shorter columns containing smaller stationary phase particles affords chiral SFC method development that is faster and more universal than previous systems. Chirality 25:799–804, 2013. © 2013 Wiley Periodicals, Inc.     

Molecular Characterization of a Genomic Fragment Containing Pi-kh Gene from the Genomic Library of Indica Rice Line Tetep

Identification of full length genes along with upstream regulatory elements is important to understand its expression. Here, we report preparation of high titre genomic library and identification of a genomic clone containing Pi-k ^h gene with its complete upstream and downstream sequences from the rice blast resistant line Tetep. Structural analysis of protein revealed that Pi-k ^h has a central nucleotide binding site domain, leucine-rich repeats domain and a unique zinc-finger domain. Comparative analysis of Pi-k ^h protein sequence showed 64% and 45% similarity with the protein sequences of rice blast resistance genes Pi-b and Pi-ta , respectively.     

Morphological and Molecular Identification of Spirometra Tapeworms (Cestoda: Diphyllobothriidae) from Carnivorous Mammals in the Serengeti and Selous Ecosystems of Tanzania

Spirometra tapeworms (Cestoda: Diphyllobothriidae) collected from carnivorous mammals in Tanzania were identified by the DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and internal transcribed spacer 1 (ITS1), and by morphological characteristics. A total of 15 adult worms were collected from stool samples and carcasses of Panthera leo, Panthera pardus, and Crocuta crocuta in the Serengeti and Selous ecosystems of Tanzania. Three Spirometra species: S. theileri, S. ranarum and S. erinaceieuropaei were identified based on morphological features. Partial cox1 sequences (400 bp) of 10 specimens were revealed. Eight specimens showed 99.5% similarity with Spirometra theileri ({"type":"entrez-nucleotide","attrs":{"text":"MK955901","term_id":"1796114604","term_text":"MK955901"}}MK955901), 1 specimen showed 99.5% similarity with the Korean S. erinaceieuropaei and 1 specimen had 99.5% similarity with Myanmar S. ranarum. Sequence homology estimates for the ITS1 region of S. theileri were 89.8% with S. erinaceieuropaei, 82.5% with S. decipiens, and 78.3% with S. ranarum; and 94.4% homology was observed between S. decipiens and S. ranarum. Phylogenetic analyses were performed with 4 species of Spirometra and 2 species of Dibothriocephalus (=Diphyllobothrium). By both ML and BI methods, cox1 and ITS1 gave well supported, congruent trees topology of S. erinaceieuropaei and S. theileri with S. decipiens and S. ranarum forming a clade. The Dibothriocephalus species were sisters of each other and collectively forming successive outgroups. Our findings confirmed that 3 Spirometra species (S. theileri, S. ranarum, and S. erinaceieuropaei) are distributed in the Serengeti and Selous ecosystems of Tanzania.     

Development of genome-wide SSR markers in horsegram and their use for genetic diversity and cross-transferability analysis

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.] commonly known as kulthi or Madras gram is an important drought tolerant legume crop used as food and fodder in India and across the globe. Horsegram is tolerant to many biotic and abiotic stresses and considered a potential future food legume. Despite being a multiutility crop, insufficient genomic information is available in this species, which is otherwise required for genetic improvement. Hence, in the present work we used next-generation sequencing (NGS) technology for genome-wide development and characterization of novel simple sequence repeat (SSR) markers in horsegram. In all, 2458 SSR primer pairs were designed from NGS data and 117 SSRs were characterized in 48 diverse lines of horsegram. Cross-transferability of these markers was also checked in nine related legume species. The polymorphic SSRs revealed high diversity measures such as mean values of expected heterozygosity (He; 0.54), observed heterozygosity (Ho; 0.64), and polymorphism information content (PIC; 0.46). Analysis of molecular variance (AMOVA) revealed high degree of genetic variance within the populations. Dendrogram based on Jaccard’s similarity coefficient and principal component analysis (PCA) revealed two groups in the analyzed accessions. This observation was further confirmed by Bayesian genetic STRUCTURE analysis. The SSR markers developed herein can be used in diverse genetic analysis including association mapping in this crop and also in related legume crops with limited marker resources. Hence, this new SSR dataset can be useful for molecular breeding research in this underutilized pulse crop. In addition, genetic diversity estimates of analyzed germplasm can be important for devising future breeding programmes in horsegram.     

Polyamines in normal and auxin-induced strawberry fruit development

The possible involvement of polyamines during strawberry ( Fragaria x ananassa Duch.) fruit development was investigated. Putrescine, spermidine, and spermine were identified in strawberry receptacles and achenes at all stages of development. Total (free) polyamine levels decreased from a maximum of 485 nmol g⁻¹ fresh weight at pollination to a minimum of 55 nmol g⁻¹ fresh weight in ripe receptacles. Total polyamine concentrations during corresponding stages of development were consistently higher in achenes than in receptacles, and ranged from 891 to 203 nmol g⁻¹ fresh weight. Removal of achenes from the surface of developing receptacles 10 days after pollination reduced receptacle growth, and re-initiation of growth by application of 1 m M α-naphtaleneacetic acid (α-NAA) was accompanied by a rapid increase in polyamine concentrations 24 h after treatment. Polyamine content per receptacle increased >3-fold in normally developing receptacles and in de-achened, auxin-treated receptacles 10 days after removal of achenes, but did not increase during this period in de-achened receptacles not treated with exogenous auxin. α-NAA increased growth and polyamine levels to a greater extent than the structurally related, but less effective auxin, β-NAA. Polyamine concentrations in receptacles with intact achenes remained similar to those of auxin depleted (de-achened) receptacles, implying that the concentration of these compounds may not be limiting following achene removal.     

Effects of satiation on feeding and swimming behaviour of planktivores

Hunger affects the feeding and swimming behaviour in fish. After 36 h of food deprivation, the feeding and swimming behaviour of Pseudorasbora parva (Cyprinidae) was studied under different prey densities (0.5, 1, 2, 5, 10 and 25 of Daphnia pulex per liter). The initial feeding rates showed marked variations in relation to prey availability. Under high prey densities, the initial feeding rate of fish was higher and subsequently decreased faster, when compared to those feeding under low prey densities. At higher prey densities, two factors were involved: that of higher prey encounter rates and also the attainment of food satiation at a faster rate. Across all prey densities, the feeding rates of fish reached a plateau after satiation. The swimming speed of fish was found to be negatively related to the prey density and a significant change in swimming speed was noted as being directly related to the level of satiation. It was found that the increasing satiation level greatly influenced the handling time and reactive volume of predator, which finally caused reduced feeding rates.     

Polyamines in normal and auxin-induced strawberry fruit development

The possible involvement of polyamines during strawberry ( Fragaria × ananassa Duch.) fruit development was investigated. Putrescine, spermidine, and spermine were identified in strawberry receptacles and achenes at all stages of development. Total (free) polyamine levels decreased from a maximum of 485 nmol g⁻¹ fresh weight at pollination to a minimum of 55 nmol g⁻¹ fresh weight in ripe receptacles. Total polyamine concentrations during corresponding stages of development were consistently higher in achenes than in receptacles, and ranged from 891 to 203 nmol g⁻¹ fresh weight. Removal of achenes from the surface of developing receptacles 10 days after pollination reduced receptacle growth, and re-initiation of growth by application of 1 m M α-naphtaleneacetic acid (α-NAA) was accompanied by a rapid increase in polyamine concentrations 24 h after treatment. Polyamine content per receptacle increased >3-fold in normally developing receptacles and in de-achened, auxin-treated receptacles 10 days after removal of achenes, but did not increase during this period in de-achened receptacles not treated with exogenous auxin. α-NAA increased growth and polyamine levels to a greater extent than the structurally related, but less effective auxin, β-NAA. Polyamine concentrations in receptacles with intact achenes remained similar to those of auxin depleted (de-achened) receptacles, implying that the concentration of these compounds may not be limiting following achene removal.