Molecular characterisation of Theileria equi and risk factors associated with the occurrence of theileriosis in horses of Punjab (Pakistan)

Theileria equi (T. equi) is an obligate intra- and extra-erythrocytic parasite that causes equine theileriosis (ET) in equids. Equine theileriosis is considered a notifiable disease of global significance, a major constraint to the international movement of horses, and endemic in many countries. This disease may be difficult to diagnose, as it can produce variable and nonspecific clinical signs. A cross-sectional study was designed for the molecular characterisation of T. equi and to investigate the associated risk factors of ET accompanied by its consequences on haematological and sero-biochemical parameters. A convenience sampling of 500 blood samples were collected from ET suspect horses from January to December 2017. PCR was performed on all blood samples targeting the 18S rRNA gene of T. equi followed by sequencing; 9% animals tested positive with confirmed sequences. The isolates of this study showed high homology with Cuban, Russian and Brazilian isolates of T. equi (accession numbers KY111762.2 , MG551915.1 and KY952237.1 , respectively). Based on multivariate analysis, the principal risk factors consisted of absence of dogs on the premises and presence of tick infestation. The haemato-biochemical parameters showed a decrease in granulocytes and erythrocytes, and an increase in lymphocytes, monocytes, mean corpuscular volume, mean corpuscular haemoglobin, mean platelet volume, glucose, phosphorus and aspartate aminotransferase in positive horses. This is the first study which identified ET in Punjab (Pakistan) using molecular techniques and risk factors together with the haemato-biochemical variations in horses.     

Molecular characterization and PCR detection of a nitrogen-fixing Pseudomonas strain promoting rice growth

Nitrogen-fixing plant growth-promoting rhizobacteria (PGPR) from the genus Pseudomonas have received little attention so far. In the present study, a nitrogen-fixing phytohormone-producing bacterial isolate from kallar grass (strain K1) was identified as Pseudomonas sp. by rrs (16S ribosomal RNA gene) sequence analysis. rrs identity level was high with an uncharacterized marine bacterium (99%), Pseudomonas sp. PCP2 (98%), uncultured bacteria (98%), and Pseudomonas alcaligenes (97%). Partial nifH gene amplified from strain K1 showed 93% and 91% sequence similarities to those of Azotobacter chroococcum and Pseudomonas stutzeri, respectively. The effect of Pseudomonas strain K1 on rice varieties Super Basmati and Basmati 385 was compared with those of three non-Pseudomonas nitrogen-fixing PGPR (Azospirillum brasilense strain Wb3, Azospirillum lipoferum strain N4 and Zoogloea strain Ky1) used as single-strain inoculants. Pseudomonas sp. K1 was detected in the rhizosphere of inoculated plants by enrichment culture in nitrogen-free growth medium, which was followed by observation under the microscope as well as by PCR using a rrs-specific primer. For both rice varieties, an increase in shoot biomass and/or grain yield over that of noninoculated control plants was recorded in each inoculated treatment. The effect of Pseudomonas strain K1 on grain yield was comparable to those of A. brasilense Wb3 and Zoogloea sp. Ky1 for both rice varieties. These results show that nitrogen-fixing pseudomonads deserve attention as potential PGPR inoculants for rice.     

The first report on serotyping of bluetongue virus in small ruminants of Khyber Pakhtunkhwa province,Pakistan

Bluetongue virus (BTV), a member of Orbivirus genus (family Reoviridae), is a non-contagious infection of domestic and wild ruminants. The current study was designed to detect various serotypes of BTV in small ruminants of Khyber Pakhtunkhwa (KPK) province of Pakistan, along with their effects on hemato-biochemical parameters. A total of 408 serum samples in four districts (Mansehra, Abbottabad, Swabi, and Kohat) of KPK from small ruminants were screened based on competitive ELISA (cELISA). A total of 204 (50%) samples were found positive for BTV group–specific antibodies. The seropositive samples were processed for the detection of BTV serotypes through real-time polymerase chain reaction (qPCR). Out of 204 cELISA-positive samples, 60 (29.41%) were found positive through qPCR. Three serotypes [6, 8, 9] were detected from Mansehra District and two from Kohat [2, 8] and Abbottabad [6, 8], while only one from Swabi [8]. The serotype “8” was found consistently in all the four study districts. A significant (p?<?0.05) increase in the level of blood urea nitrogen (BUN) and alkaline phosphatase (ALP) was recorded in goats, whereas aspartate aminotransferase (AST) in sheep infected with BTV, compared to healthy animals. The hematological parameters showed significantly (p?<?0.05) raised total leucocyte count (TLC) in both sheep and goats, whereas only hematocrit (HCT) value was increased significantly (p?<?0.05) in infected sheep. This is the first report on serotyping of BTV among small ruminants in Pakistan.

     

Influence of organic and inorganic passivators on Cd and Pb stabilization and microbial biomass in a contaminated paddy soil

Purpose

Soil contamination with heavy metals, such as Cd and Pb, has caused severe health and environmental risks all over the world. Possible eco-friendly solutions for Cd and Pb immobilization were required to reduce its mobility through various cost-effective amendments.

Materials and methods

A laboratory incubation study was conducted to assess the efficiency of biochar (BC), zeolite (ZE), and rock phosphate (RP) as passivators for the stabilization of Cd and Pb in paddy soil as well as soil microbial biomass. Various extraction techniques were carried out: a sequential extraction procedure, the European Community Bureau of Reference (BCR), toxicity characteristic leaching procedure (TCLP) test, and single extraction with CaCl₂. The impact of passivators on soil pH, dissolved organic carbon (DOC), and microbial biomass (carbon, nitrogen, and phosphorus) was examined in the metal contaminated soil.

Results and discussion

The results showed that the exchangeable portion of Cd in soil was significantly reduced by 34.8, 21.6, and 18.8% with ZE, RP, and BC at a 3% application rate, respectively. A similar tendency of reduction in Pb soluble portion was observed by ZE (9.6%), RP (20%), and BC (21.4%) at a 3% application rate. Moreover, the TCLP leachate of Cd and Pb was apparently reduced by 17 and 30.3% with BC at a 3% application dose, respectively, when compared to the control. Soil pH, nutrients, and microbial biomass C, N, and P were significantly increased with the addition of BC, RP, and ZE passivators.

Conclusions

The results showed that the incorporation of BC, ZE, and RP significantly reduced the Cd and Pb mobility in paddy soil as well as enhanced soil nutrients and microbial biomass. Overall, among all the amendments, rice straw derived-BC performed better for Cd and Pb immobilization in paddy soil.

     

Comparative Efficacy of CO2 and Ozone Gases Against Ephestia cautella (Lepidoptera: Pyralidae) Larvae Under Different Temperature Regimes

Comparative efficacy of three different modified atmospheres: 100% CO₂, 75% CO₂ + 25% N₂, and 22 ppm ozone were examined against larval mortality of the almond moth, Ephestia cautella (Walker) (Lepidoptera: Pyralidae) at temperature regimes of 25°C and 35 ± 2°C and 60 ± 5% relative humidity, and 9:15 dark and light. Wandering young larval instars, which are fast growing, large enough in size and considered as more tolerant to modified atmosphere, were collected directly from the rearing culture, placed inside pitted date fruits of vars.: “Khudri,” “Ruziz,” and “Saqie,” were treated with aforementioned gases for 24, 48, and 72 h. The immediate and delayed larval mortality was recorded after each exposure timing. Ozone possessed the strongest fumigant toxicity causing 100% mortality with all varieties, at 25 and 35°C after 24 h exposure and was more effective than 75% CO₂ that caused 83 and 100% immediate mortality with variety ruziz at 25 and 35°C, respectively. Extending the treatments exposure time to 72 h, 100% mortality was recorded by exposing larvae to any of the studied gases at 25 and 35°C. These results suggest that gases and temperature used in this study can be effectively used to control E. cautella in dates and stored grains.     

Tree age influences nutritional,pectin, and anatomical changes in developing ‘Kinnow’ mandarin (Citrus nobilis Lour × Citrus deliciosa Tenora) fruit

An understanding of variation in fruit quality in relation to tree age can help understand the issue of poor fruit quality in young orchards; however, limited information is available on the citrus fruit. In this study, the endogenous nutritional status in rind, rag, and leaves; pectin status in rind and rag; and anatomical fruit growth parameter in rind tissues were studied in ‘Kinnow’ mandarins fruit during their development on trees from three age (6-, 18-, and 35-year-old) groups. In older (35-year-old) trees, rind, rag, and leaf nitrogen (N), phosphorus (P), and potassium (K) concentrations were superior. In fruit from all tree age groups, total pectin and protopectin reduced; however water-soluble pectin (WSP) improved. In rind tissues harvested from young (6-year-old) trees, cell density was more while cell size was less. In all tree age groups, cell density in rind tissues correlated negatively with rind WSP.     

Influence of nitrogen fertilizer forms and crop straw biochars on soil exchange properties and maize growth on an acidic Ultisol

Effects of repeated application of urea (UN) and calcium nitrate (CN) singly and together with crop straw biochars on soil acidity and maize growth were investigated with greenhouse pot experiments for two consecutive seasons. Canola straw biochar (CB), peanut straw biochar (PB) and wheat straw biochar (WB) were applied at 1% of dried soil weight in the first season. N fertilizers were applied at 200 mg N kg^?1. In UN treatments, an initial rise in pH was subjected to proton consumption through urea hydrolysis, afterwards nitrification of NH₄⁺ caused drastic reductions in pH as single UN had soil pH of 3.70, even lower than control (4.27) after the 2nd crop season. Post-harvest soil analyses indicated that soil pH, soil exchangeable acidity, NH₄⁺, NO₃^? and total base cations showed highly significant variation under N and biochar types (P < 0.05). Articulated growth of plants under combined application with biochars was expressed by 22.7%, 22.5%, and 35.7% higher root and 25.6%, 23.8%, and 35.9% higher shoot biomass by CB, PB and WB combined with CN over UN, respectively. Therefore, CN combined with biochars is a better choice to correct soil acidity and improve maize growth than UN combined with biochars.     

Estimation of economic threshold level of alligator weed (Alternanthera philoxeroides (Mart.) Griseb.) to Tackle Grain quality and yield Losses in Rice

Alligator weed (Alternanthera philoxeroides) is the most troublesome invasive weed in transplanted rice ecosystems worldwide. A two-year field study was conducted to estimate economic threshold levels and the season long competitive effect of six alligator weed densities (0, 2, 4, 8, 16, and 32 plants m^?2) on yield and quality traits of transplanted rice. A gradual linear incline in dry weight of alligator weed was observed with an increase in its density from 2 to 32 plants m^?2. Maximum N (15.93–18.26 kg ha^?1), P (15.10–16.46 kg ha^?1) and K (16.34–17.81 kg ha^?1) uptake by alligator weed was recorded at its density of 32 plants m^?2. More micronutrient loss to the tune of 6.53, 47.92, 19.99, and 38.42 mg kg^?1 for Cu, Fe, Zn, and Mn, respectively was observed at the same density. Increasing density of alligator weed caused more losses in paddy yield (up to 21.37–23.78%), amylose and rice grain protein contents. According to a nonlinear hyperbolic regression model, maximum paddy yield loss of rice at asymptotic value of alligator weed density was 38.8% during 2011 and 42.4% during 2012. Economic threshold value of alligator weed was estimated 1.5 and 1.3 plants per m² during 2011 and 2012, respectively.     

Molecular and biologic characterization of a phytoplasma associated with <Emphasis Type="Italic">Brassica campestris</Emphasis> phyllody disease in Punjab province,Pakistan

A phytoplasma-associated disease was identified in Brassica campestris (sarson) plants during a survey conducted in Punjab province of Pakistan in 2014–2016. The symptomatic plants showed characteristic symptoms of phyllody and witches’ broom. Phytoplasma presence was detected by polymerase chain reaction on 16S ribosomal and tuf DNAs, followed by RFLP analysis and sequence comparison of the 16S rRNA and tuf genes. The phytoplasma detected was classified in a new ribosomal subgroup designed 16SrIX-H. The phytoplasma presence in phloem tissues of symptomatic sarson samples was also confirmed through light microscopy and transmission studies to healthy plants through dodder and the leafhopper Orosius albicinctus. This is the first report of identification of 16SrIX-H subgroup phytoplasma associated with sarson and its natural vector in Pakistan.