Muhammad Saleem Haider

Ageratum enation virus—A Begomovirus of Weeds with the Potential to Infect Crops

Samples of two Ageratum conyzoides, one Sonchus oleraceus and one turnip (Brassica rapa var. rapa) exhibiting virus-like symptoms were collected from Pakistan and Nepal. Full-length begomovirus clones were obtained from the four plant samples and betasatellite clones from three of these. The begomovirus sequences were shown to be isolates of Ageratum enation virus (AEV) with greater than 89.1% nucleotide sequence identity to the 26 AEV sequences available in the databases. The three betasatellite sequences were shown to be isolates of Ageratum yellow leaf curl betasatellite (AYLCB) with greater than 90% identity to the 18 AYLCB sequences available in the databases. The AEV sequences were shown to fall into two distinct strains, for which the names Nepal (consisting of isolates from Nepal, India, and Pakistan—including the isolates identified here) and India (isolates occurring only in India) strains are proposed. For the clones obtained from two AEV isolates, with their AYLCB, infectivity was shown by Agrobacterium-mediated inoculation to Nicotiana benthamiana, N. tabacum, Solanum lycopersicon and A. conyzoides. N. benthamiana plants infected with AEV alone or betasatellite alone showed no symptoms. N. benthamiana plants infected with AEV with its associated betasatellite showed leaf curl symptoms. The findings show that AEV is predominantly a virus of weeds that has the capacity to infect crops. AYLCB appears to be the common partner betasatellite of AEV and is associated with diseases with a range of very different symptoms in the same plant species. The inability to satisfy Koch’s postulates with the cloned components of isolate SOL in A. conyzoides suggests that the etiology may be more complex than a single virus with a single betasatellite.

Novel Maltotriose-Hydrolyzing Thermoacidophilic Type III Pullulan Hydrolase from Thermococcus kodakarensis

ABSTRACT A novel thermoacidophilic pullulan-hydrolyzing enzyme (PUL) from hyperthermophilic archaeon Thermococcus kodakarensis (TK-PUL) that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions was cloned and characterized. TK-PUL possessed both pullulanase and α-amylase activities. The highest activities were observed at 95 to 100°C. Although the enzyme was active over a broad pH range (3.0 to 8.5), the pH optima for both activities were 3.5 in acetate buffer and 4.2 in citrate buffer. TK-PUL was stable for several hours at 90°C. Its half-life at 100°C was 45 min when incubated either at pH 6.5 or 8.5. The Km value toward pullulan was 2 mg ml−1, with a V max of 109 U mg−1. Metal ions were not required for the activity and stability of recombinant TK-PUL. The enzyme was able to hydrolyze both α-1,6 and α-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was γ-cyclodextrin, which is a novel feature for this type of enzyme. Additionally, the enzyme hydrolyzed a variety of polysaccharides, including starch, glycogen, dextrin, amylose, amylopectin, and cyclodextrins (α, β, and γ), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.

Genetics and mechanism of resistance to deltamethrin in the house fly, Musca domestica L., from Pakistan

Deltamethrin (a pyrethroid insecticide) has widely been used against the house fly, Musca domestica, a pest found in livestock facilities worldwide. Although, cases of both metabolic and physiological resistance to deltamethrin have been reported in different parts of the world, no studies have been reported to characterize this resistance in house flies from Pakistan. In the present study, we investigated a field strain of house flies for potential to develop resistance to deltamethrin. Also, its stability, possible mechanisms and cross-resistance potential to other insecticides. Before the selection experiments, the field strain showed 8.41-, 3.65-, 8.39-, 2.68-, 19.17- and 5.96-fold resistance to deltamethrin, bifenthrin, lambda-cyhalothrin, chlorpyrifos, profenofos and spinosad, respectively, compared with the reference strain (Lab-susceptible). Continuous selection of the field strain (Delta-SEL) with deltamethrin for six generations (G1–G6) in the laboratory increased the resistance ratio to 176.34 after bioassay at G7. The Delta-SEL strain was reared for the next four generations without exposure to deltamethrin and bioassayed at G11 which revealed that the resistance was stable. The Delta-SEL strain at G7 showed cross-resistance to all other insecticides except spinosad, when compared to the bioassays before the selection experiment (G1). Crosses between Delta-SEL and Lab-susceptible strains revealed an autosomal and incomplete dominant mode of resistance to deltamethrin. A direct test using a monogenic inheritance model revealed that the resistance was governed by more than one factor. Moreover, synergism studies with the enzyme inhibitors PBO and DEF reduced the resistance to deltamethrin in the selected strain up to 2.51- and 2.19-fold, respectively, which revealed that the resistance was possibly due to microsomal oxidase and esterase activity. It is concluded that the resistance to deltamethrin was autosomal and incompletely dominant. The high cross-resistance of bifenthrin, lambda-cyhalothrin, chlorpyrifos and profenofos in the Delta-SEL strain suggests that other insecticides would be necessary to counter the resistance. These results are therefore suggestive for implications in the management of insecticide resistance in house flies.

Chili leaf curl betasatellite is associated with a distinct recombinant begomovirus, Pepper leaf curl Lahore virus, in Capsicum in Pakistan

Capsium spp. are an important vegetable crop cultivated through Pakistan. Leaf curl disease is the major disease of Capsicum spp. in Pakistan caused by viruses. The disease has previously been shown to be associated with begomoviruses and betasatellites. We have cloned and sequenced a begomovirus and its associated betasatellite from Capsicum originating from central Pakistan. The begomovirus isolated was distinct from all previously characterised viruses and we propose the name Pepper leaf curl Lahore virus (PepLCLV) for this new species. Comparison of the sequence of PepLCLV with previously characterised begomoviruses shows it likely to have resulted from recombination between Papaya leaf curl virus and Chili leaf curl virus (ChiLCV), two species that have previously been identified in Pakistan. The betasatellite associated with PepLCLV in Capsicum was identified as Chili leaf curl betasatellite (ChLCB). This is the first identification of a cognate begomovirus for ChLCB infecting Capsicum, although this betasatellite has been shown in association with ChiLCV infecting potato in Pakistan. PepLCLV is one of an increasing number of monopartite begomoviruses shown to be associated with a betasatellite and one of the numerous species that affect Capsicum. In view of their only having been identified in Pakistan, PepLCLV and ChLCB likely represent a geographically distinct, Capsicum adapted, begomovirus-betasatellite complex.

First report of Squash leaf curl China virus in Pakistan

Leaf samples of Cucurbita pepo plants showing yellow mosaic symptoms were collected from Lahore, Pakistan. Full-length clones of a bipartite begomovirus were isolated and sequenced in their entirety. The sequences showed the highest levels of nucleotide sequence identity (98.4% for the DNA-A and 89.6% for the DNA-B) to the ‘Indian’ strain of Squash leaf curl China virus (SLCCNV).

Breeding potential of the basmati rice germplasm under water stress condition

Eight parents were selected on the basis of phenotypic and genotypic screening for the development of F 1 . All the possible combinations were made between the parents excluding reciprocals in diallel mating design. Data were analyzed by using Hayman graphical approach and Griffing’s approach to study the genetics of the parents and their F 1 hybrids. Based on the genetic component analysis, both additive and non-additive components appeared which is important in the inheritance of most of the traits. Both additive and dominance type of gene action were found important in inheritance for different traits under study. Most of the traits showed constant gene action in both environments, but the gene action of some traits was affected by the environment. Morphological traits like plant height, productive tillers per plant and 1000 seed weight showed over dominance type of gene action in both environments (control and drought environments), while seeds per panicle and seed length width ratio showed this type of gene action only in drought conditions. The seeds per panicle and length width ratio showed additive type of gene action with partial dominance only in normal irrigation conditions. From Griffing analysis, genotypes CB-17, CB-32 and Basmati-198 were found to be good general combiners for productive tillers per plant, primary branches per panicle and yield per plant, especially under water stress condition. Also, maximum specific combining ability was found in Basmati-198 × CB-17 for productive tillers per plant, Basmati-198 × CB-42 for primary branches per panicle and CB-32 × CB-14 for yield per plant. Key words: Oryza sativa L., gene action, combining ability, stress, yield traits.

Impact of copper toxicity on stone-head cabbage (Brassica oleracea var. capitata) in hydroponics

Arable soils are frequently subjected to contamination with copper as the consequence of imbalanced fertilization with manure and organic fertilizers and/or extensive use of copper-containing fungicides. In the present study, the exposure of stone-head cabbage (Brassica oleracea var. capitata) to elevated Cu2+ levels resulted in leaf chlorosis and lesser biomass yield at ≥2 µ M. Root nitrate content was not statistically affected by Cu2+ levels, although it was substantially decreased at ≥5 µ M Cu2+ in the shoot. The decrease in nitrate contents can be related to lower nitrate uptake rates because of growth inhibition by Cu-toxicity. Shoot sulfate content increased strongly at ≥2 µ M Cu2+ indicating an increase in demand for sulfur under Cu stress. Furthermore, at ≥2 µM concentration, concentration of water-soluble non-protein thiol increased markedly in the roots and to a smaller level in the shoot. When exposed to elevated concentrations of Cu2+ the improved sulfate and water-soluble non-protein thiols need further studies for the evaluation of their direct relation with the synthesis of metal-chelating compounds (i.e., phytochelatins).

Risk assessment, cross-resistance potential, and biochemical mechanism of resistance to emamectin benzoate in a field strain of house fly (Musca domestica Linnaeus)

Reduced sensitivity to insecticides in insect pests often results in control failures and increases in the dose and frequency of applications, ultimately polluting the environment. Reduced sensitivity to emamectin benzoate, a broad-spectrum agrochemical belonging to the avermectin group of pesticides, was reported in house flies (Musca domestica L.) collected from Punjab, Pakistan, in 2013. The aim of the present study was to investigate the risk for resistance development, biochemical mechanism, and cross-resistance potential to other insecticides in an emamectin benzoate selected (EB-SEL) strain of house flies. A field-collected strain showing reduced sensitivity to emamectin was re-selected in the laboratory for five consecutive generations and compared with a laboratory susceptible (Lab-Susceptible) reference strain, using bioassays. The field strain showed rapid development of resistance to emamectin (resistance ratio (RR) increased from 35.15 to 149.26-fold) as a result of selection experiments; however, resistance declined when the selection pressure uplifted. The EB-SEL strain showed reduction in resistance to abamectin, indoxacarb, and thiamethoxam. The results of synergism experiments using piperonyl butoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) enzyme inhibitors and biochemical analyses revealed that the metabolic resistance mechanism was not responsible in developing emamectin resistance in the EB-SEL strain. In conclusion, the risk for the rapid development of emamectin resistance under continuous selection pressure suggests using a multifaceted integrated pest management approach for house flies. Moreover, the instable nature of emamectin resistance in the EB-SEL strain and lack of cross-resistance to other insecticides provide windows for the rotational use of insecticides with different modes of action. This will ultimately reduce emamectin selection pressure and help improving management programs for house flies without polluting the environment.

First report of Cochliobolus australiensis causing leaf spot of bermudagrass in Pakistan.

Bermuda grass (Cynodon dactylon) plants showing symptoms of light brown to blackish leaf spots were observed during spring 2012 in the parks and grassy lawns of Lahore (Pakistan). Samples from infected plants were collected and a fungus consistently isolated on potato dextrose agar (PDA) at 28±1°C. The fungus was identified as Bipolaris dactylon (telomorph Cochliobolus australiensis) based on its morphological and molecular features. The pathogen identity was verified by the First Fungal Culture Bank of Pakistan (FCBP) and a culture deposited (accession No. FCBP-1288). In addition, the fungal DNA was isolated and the internally transcribed spacer (ITS) region amplified (White et al., 1990), cloned, sequenced and deposited in GenBank (accession No. HE962033). Sequencing of the ITS region of ribosomal DNA showed a 99.8% nucleotide sequence identity with C. australiensis isolates from Texas (HQ608034) and India (AY923860), respectively. Reproduction of symptoms in inoculated healthy plants fulfilled Koch’s postulates and confirmed pathogenicity. B. australiensis had previously been isolated from infected roots of C. dactylon during a survey in Pakistan (Shahzad and Ghaffar, 1995). However, no details were given of foliar symptoms, nor any pathogenicity test was conducted. Therefore, to the best or our knowledge, this is the first confirmed report of B. dactylon causing a leaf spot on Bermudagrass in Pakistan.

Complete nucleotide sequences of a distinct bipartite begomovirus, bitter gourd yellow vein virus, infecting Momordica charantia

Momordica charantia (Cucurbitaceae), a vegetable crop commonly cultivated throughout Pakistan, and begomoviruses, a serious threat to crop plants, are natives of tropical and subtropical regions of the world. Leaf samples of M. charantia with yellow vein symptoms typical of begomovirus infections and samples from apparently healthy plants were collected from areas around Lahore in 2004. Full-length clones of a bipartite begomovirus were isolated from symptomatic samples. The complete nucleotide sequences of the components of one isolate were determined, and these showed the arrangement of genes typical of Old World begomoviruses. The complete nucleotides sequence of DNA A showed the highest nucleotide sequence identity (86.9%) to an isolate of Tomato leaf curl New Delhi virus (ToLCNDV), confirming it to belong to a distinct species of begomovirus, for which the name Bitter gourd yellow vein virus (BGYVV) is proposed. Sequence comparisons showed that BGYVV likely emerged as a result of inter-specific recombination between ToLCNDV and tomato leaf curl Bangladesh virus (ToLCBDV). The complete nucleotide sequence of DNA B showed 97.2% nucleotide sequence identity to that of an Indian strain of Squash leaf curl China virus.