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.

A leaf spot of rice (Oryza sativa L.) was observed in a survey conducted in different districts of Punjab, Pakistan, from August to October, 2012. Symptoms appeared as brown spots on the leaf blade and on the small emerging leaves. Primary symptoms were brown small, round-ovoid spots with a chlorotic halo, evenly distributed over the leaf. The size of spots varies from 10mm – 1 cm. Over time, spots merged and became large. Infected leaves were collected and washed in running water thoroughly for 5-10 min. Small pieces (0.5-2 cm) of leaf tissue were then surface disinfected with 2% NaOCl for 2 min, rinsed three to four times in sterile distilled water (SDW), blotted dry on sterile filter paper and placed on potato dextrose agar (PDA) plates. PDA plates were incubated at 28 0C for 5-7 days. A single spore fungal isolate was maintained on PDA for further investigations. Morphological and microscopic characteristics and phylogenetic analysis were used for the identification for the fungal isolate. On PDA, fun...