cA 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 ␣-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 K m 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 ␣-1,6 and ␣-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was ␥-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 (␣, ␤, and ␥), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.

As part of a study to determine the diversity of whitefly-transmitted viruses (genus Begomovirus, family Geminiviridae) associated with cotton leaf curl disease in Pakistan, leaf samples from cotton plants showing typical leaf curl disease symptoms were collected in various locations of Punjab province. Sequence analysis of full-length virus clones (~2.7 kb) showed plants to be infected with the begomovirus cotton leaf curl Burewala virus, the only virus identified in cotton in the Punjab since 2001. Surprisingly, a second virus, the leafhopper-transmitted chickpea chlorotic dwarf virus (CpCDV) of the genus Mastrevirus (family Geminiviridae), was identified in a small number of plants. The sequences of four CpCDV isolates from cotton originating from geographically distinct areas in Punjab were obtained. Analysis of the sequences showed them to represent a distinct, newly identified strain of CpCDV with the highest levels of nucleotide sequence identity to isolates of CpCDV strains C and D that have been identified previously in Pakistan. CpCDV has not been identified previously in cotton. The significance of this finding is discussed.

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The individual role of biochar, compost and PGPR has been widely studied in increasing the productivity of plants by inducing resistance against phyto-pathogens. However, the knowledge on combined effect of biochar and PGPR on plant health and management of foliar pathogens is still at juvenile stage. The effect of green waste biochar (GWB) and wood biochar (WB), together with compost (Comp) and plant growth promoting rhizobacteria (PGPR; Bacillus subtilis) was examined on tomato (Solanum lycopersicum L.) physiology and Alternaria solani development both in vivo and in vitro. Tomato plants were raised in potting mixture modified with only compost (Comp) at application rate of 20% (v/v), and along with WB and GWB at application rate of 3 and 6% (v/v), each separately, in combination with or without B. subtilis. In comparison with WB amended soil substrate, percentage disease index was significantly reduced in GWB amended treatments (Comp + 6%GWB and Comp + 3%GWB; 48.21 and 35.6%, respectively). Whereas, in the presence of B. subtilis disease suppression was also maximum (up to 80%) in the substrate containing GWB. Tomato plant growth and physiological parameters were significantly higher in treatment containing GWB (6%) alone as well as in combination with PGPR. Alternaria solani mycelial growth inhibition was less than 50% in comp, WB and GWB amended growth media, whereas B. subtilis induced maximum inhibition (55.75%). Conclusively, the variable impact of WB, GWB and subsequently their concentrations in the soil substrate was evident on early blight development and plant physiology. To our knowledge, this is the first report implying biochar in synergism with PGPR to hinder the early blight development in tomatoes.

Bitter gourd ( Momordica charantia ; family Cucurbitaceae) is a vegetable cultivated in many areas of Pakistan. Plants showing yellow blotch symptoms were observed in several fields in the vicinity of Lahore, Pakistan, with an average incidence of 60 -70%. Leaf samples were collected from four diseased and from two apparently healthy (symptomless) plants, and used for DNA extraction.The presence of a begomovirus was confirmed by PCR amplification using a degenerate primer pair designed to conserved regions of the coat protein genes from published sequences of begomoviruses from the Old World (Haider, 1996; virion-An amplification product of the expected size ( ≈ 750 bp) was produced from samples from plants with symptoms, but not from symptomless plants. The PCR product was cloned and sequenced (accession no. AJ854186). The sequence showed the highest levels of sequence identity (95%) to Tomato leaf curl New Delhi virus (ToLCNDV; U15015), indicating that the virus of M. charantia is a strain of ToLCNDV.Recent reports indicate that many Old World begomoviruses are associated with a single-stranded DNA satellite (DNA-β ;Briddon et al. , 2003). Attempts to identify the presence of a DNA-β in infected M. charantia using universal DNA-β primers (Briddon et al ., 2002) produced a 0·6 kb band that may be a defective DNA-β . Efforts are now under way to sequence this and to produce full-length clone(s) of the virus. This is the first report of ToLCNDV infecting M. charantia .