K. A. Malik

Cotton leaf curl disease is associated with multiple monopartite begomoviruses supported by single DNA β

Summary For bipartite begomoviruses (family Geminiviridae) trans-replication of the DNA B component by the DNA A-encoded replication-associated protein (Rep) is achieved by virtue of a shared sequence, the “common region”, which contains repeated motifs (iterons) which are sequence-specific Rep binding sites and form part of the origin of replication. Recently cotton leaf curl disease (CLCuD), a major constraint to cotton production on the Indian subcontinent, has been shown to be caused by a monopartite begomovirus (Cotton leaf curl Multan virus [CLCuMV]) and a novel single-stranded DNA satellite molecule termed CLCuD DNA β. The satellite molecule is trans-replicated by CLCuMV but does not possess the iteron sequences of this virus. We have investigated the ability of CLCuD DNA β to interact with three further clones of monopartite begomoviruses, isolated from cotton, that have distinct Rep binding specificities. All three cloned viruses were capable of trans-replicating the satellite molecule and inducing CLCuD symptoms in cotton, indicating that the interaction between begomovirus and DNA β is relaxed in comparison to the interaction between DNA A and DNA B components. Field surveys across all the cotton growing regions of Pakistan indicate that dual and multiple infections are the norm for CLCuD with no evidence of synergism. Despite the diversity of begomoviruses associated with CLCuD, only a single class of DNA β has been detected, suggesting that this satellite has the capacity to be recruited by unrelated begomoviruses.

Genetic diversity evaluation of some elite cotton varieties by RAPD analysis.

Random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity of elite commercial cotton varieties. Twenty two varieties belonging to Gossypium hirsutum L. and one to G. arboreum L. were analyzed with 50 random decamer primers using the polymerase chain reaction (PCR). Forty nine primers detected polymorphism in all 23 cotton varieties, while one produced monomorphic amplification profiles. A total of 349 bands were amplified, 89.1% of which were polymorphic. Cluster analysis by the unweighted pair group method of arithmetic means (UPGMA) showed that 17 varieties can be placed in two groups with a similarity ranging from 81.51% to 93.41%. G. hirsutum L. varieties S-12, V3 and MNH-93 showed a similarity of 78.12, 74.46 and 69.56% respectively with rest of the varieties. One variety, CIM-1100, showed 57.02% similarity and was quite distinct. The diploid cotton G. arboreum L. var. Ravi was also very distinct from rest of its tetraploid counterparts and showed only 55.7% similarity. The analysis revealed that the intervarietal genetic relationships of several varieties is related to their center of origin. As expected, most of the varieties have a narrow genetic base. The results obtained can be used for the selection of possible parents to generate a mapping population. The results also reveal the genetic relationship of elite commercial cotton varieties with some standard “Coker” varieties and the diploid G. arboreum L. var. Ravi (old world cotton).

Association of nitrogen-fixing, plant-growth-promoting rhizobacteria (PGPR) with kallar grass and rice

Leptochloa fusca (L.) Kunth (kallar grass) has previously been found to exhibit high rates of nitrogen fixation. A series of experiments to determine the level of biological nitrogen fixation using 15N isotopic dilution were carried out in nutrient solution and saline soil. These studies indicated an agronomically significant amount of nitrogen being fixed in soil. Kallar grass has a similar growth habitat to rice. Therefore similar studies were carried out with rice after isolating various diazotrophs from the roots which were also screened for their ability to produce auxin (IAA). Five such strains namely Azospirillum lipoferum N-4, Azospirillum brasilense Wb-3, Azoarcus K-1, Pseudomonas 96–51, Zoogloea Ky-1 were selected for inoculating two rice varieties i.e. NIAB-6 and BAS-370 under aseptic laboratory conditions. The nitrogen fixed was quantified using the 15N isotopic dilution method. Variety BAS-370 had nearly 70% nitrogen derived from atmosphere (Ndfa) when inoculated with Azospirillum N-4. Similar studies with the mixed inoculum using 15N fertilizer in the micro plots indicated that nearly 29% of plant nitrogen was derived from the atmosphere.

Transgenic tobacco expressing geminiviral RNAs are resistant to the serious viral pathogen causing cotton leaf curl disease

Summary. Cotton, the major cash crop in Pakistan, suffers 30% losses to cotton leaf curl disease, caused by the geminivirus, cotton leaf curl virus DNA A, plus a satellite component, DNA β responsible for symptom development with plants failing to produce cotton bolls. We constructed transgenic tobacco expressing sense and antisense RNAs representing: [i] the 5′ half of the viral DNA replication gene, AC1, [ii] the 3′ half of AC1, [iii] two overlapping genes, AC2, a transcription activator, and AC3, a replication enhancer. In contrast to controls, 25% of 72 transgenic tobacco lines tested showed heritable resistance [T1 − T3 generations]: symptom-free and no replication of DNA A or DNA β even after 120 days of continuous exposure to viruliferous whiteflies. As geminiviral and transgene RNAs are not detected in resistant lines following infection, and selected uninfected resistant tobacco sense lines reveal double-stranded and small interfering RNAs, the most likely mechanism is via post-transcriptional gene silencing.

Cellulase production by Cellulomonas biazotea cultured in media containing different cellulosic substrates

Abstract Production of filter paper cellulase (FPase), endo-β-glucanase and β-glucosidase by Cellulomonas biazotea was investigated during growth on different substrates. The organism utilized four different cellulosics, NaOH-pretreated ground plant material of four lignocellulosic (LC) substrates grown on saline lands, three agricultural wastes, carboxymethyl cellulose (CMC), cellobiose and xylan as carbon sources in Dubos salts liquid medium and produced the enzymes. The highest level of volumetric productivity ( Q p ) of FPase occurred in the cell-free supernatants of C. biazotea during growth on α-cellulose followed by Leptochloa fusca (kallar grass), while that of endo-β-glucanase occurred on kallar grass followed by α-cellulose. Maximum β-glucosidase was produced in culture media containing cellobiose and kallar grass as carbon sources. Thus the production of these enzymes is influenced by the carbon source used. β-Glucosidase was produced mainly periplasmic and was several fold greater in quantity than that reported in other strains of Cellulomonas , as well as other bacteria. Kallar grass culture medium, during growth of C. biazotea , supported maximum Q p levels of 37.5, 17.5 and 6.1 IU/l/h for CMCase, FPase and β-glucosidase, respectively, with cell mass productivity of 0.235 g/l/h and was selected as a preferred substrate for cellulase production.

Introduction: Assessing opportunities for nitrogen fixation in rice — a frontier project

Recent advances in understanding symbiotic Rhizobium-legume interactions at the molecular level, the discovery of endophytic interactions of nitrogen-fixing organisms with non-legumes, and the ability to introduce genes into rice by transformation have stimulated researchers world wide to harness opportunities for nitrogen fixation and improved N nutrition in rice. In a think-tank workshop organized by IRRT in 1992, the participants reaffirmed that such opportunities do exist for cereals and recommended that rice be used as a model system. Subsequently, IRRI developed a New Frontier Project to coordinate the worldwide collaborative efforts among research centers committed to reducing dependency of rice on mineral N resources. An international Biological Nitrogen Fixation (BNF) working group was established to review, share research results/materials and to catalyze research.

Molecular phylogeny of Gossypium species by DNA fingerprinting

Abstract Total genomic DNA from 31 available Gossypium species, three subspecies and one interspecific hybrid, were analysed to evaluate genetic diversity by RAPD, using 45 random decamer primers. A total of 579 amplified bands were observed, with 12.9 bands per primer, of which 99.8% were polymorphic. OPJ-17 produced the maximum number of fragments while the minimum number of fragments was produced with primer OPA-08. Cluster analysis by the unweighted paired group method of arithmetic means (UPGMA) showed six main clusters. Cluster ’A’ consisted of two species and one subspecies of the A-genome, with a 0.78–0.92 Nei’s similarity range. Cluster B, composed of all available tetraploid species and one interspecific hybrid, showed the same sister cluster. Nei’s similarity ranged from 0.69 to 0.84. The B-genome formed the UPGMA sister cluster to the E-genome species. Cluster ’C’ consisted of five Gossypium species of which three belong to the B-genome, with Nei’s similarity values of 0.81 to 0.86. Although there was considerable disagreement at lower infra-generic ranks, particularly among the D- genome (diploid New World species) and C-genome (diploid Australian species) species. The sole F-genome species Gossypium longicalyx was resolved as a sister group to the D-genome species. Gossypium herbaceum and G. herbaceum Africanum showed the maximum Nei’s similarity (0.93). Minimum similarity (0.29) was observed between Gossypium trilobum and Gossypium nelsonii. The average similarity among all studied species was 50%. The analysis revealed that the interspecific genetic relationship of several species is related to their centre of origin. As expected, most of the species have a wide genetic base range. The results also revealed the genetic relationships of the species Gossypium hirsutum to standard cultivated Gossypium barbadense, G. herbaceum and Gossypium arboreum. These results correspond well with previous reported results. The level of variation detected in closely related genotypes by RAPD analysis indicates that it may be a more efficient marker than morphological marker, isozyme and RFLP technology for the construction of genetic linkage maps.

Rhizobial inoculation improves seedling emergence, nutrient uptake and growth of cotton

Experiments were conducted to determine the growth promoting activities of various rhizobia in cotton (Gossypium hirsutum L.) under growth room conditions. Seeds of 4 cotton cultivars were inoculated with 4-indole-3-acetic acid producing selected (Brady) rhizobium strains and Azotobacter plant growth promoting rhizobacteria strains, included as a positive control. Growth responses to inoculation exhibited bacterial strain-cotton cultivar specificity and also included increase in rate of seedling emergence by 3–9%. Shoot dry weight, biomass and N uptake were increased by 48, 75 and 57%, respectively, due to inoculation with both the Rhizobium leguminosarum bv. trifolii E11 and Azotobacter sp. S8, whereas, strain E11 also increased root dry weight, root length and area by 248, 332 and 283%, respectively. K+ and Ca2+ uptake was also increased by 2–21% and 9–14%, respectively, due to rhizobial inoculation. The results also showed that (Brady) rhizobium strains promoted cotton growth through efficient nutrient uptake, which was mainly related to increased root growth due to the effect of IAA produced by these strains. However, growth promotion by Azotobacter sp. S8, in addition to 4-indole-3-acetic acid production, might also involve biological N2 fixation by this rhizobacterial strain at some stage during its growth.

Rhizobium, Bradyrhizobium and Agrobacterium strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.

Nitrous oxide emissions from an irrigated sandy-clay loam cropped to maize and wheat

Abstract Nitrous oxide (N2O) emissions were measured from an irrigated sandy-clay loam cropped to maize and wheat, each receiving urea at 100 kg N ha–1. During the maize season (24 August–26 October), N2O emissions ranged between –0.94 and 1.53 g N ha–1 h–1 with peaks during different irrigation cycles (four) ranging between 0.08 and 1.53 g N ha–1 h–1. N2O sink activity during the maize season was recorded on 10 of the 29 sampling occasions and ranged between 0.18 and 0.94 g N ha–1 h–1. N2O emissions during the wheat season (22 November–20 April) varied between –0.85 and 3.27 g N ha–1 h–1, whereas peaks during different irrigation cycles (six) were in the range of 0.05–3.27 g N ha–1 h–1. N2O sink activity was recorded on 14 of the 41 samplings during the wheat season and ranged between 0.01 and 0.87 g N ha–1 h–1. Total N2O emissions were 0.16 and 0.49 kg N ha–1, whereas the total N2O sink activity was 0.04 and 0.06 kg N ha–1 during the maize and wheat seasons, respectively. N2O emissions under maize were significantly correlated with denitrification rate and soil NO3–-N but not with soil NH4+-N or soil temperature. Under wheat, however, N2O emissions showed a strong correlation with soil NH4+-N, soil NO3–-N and soil temperature but not with the denitrification rate. Under either crop, N2O emissions did not show a significant relationship with water-filled pore space or soil respiration.