Khalid Pervaiz Akhtar

Influence of plant age, whitefly population and cultivar resistance on infection of cotton plants by cotton leaf curl virus (CLCuV) in Pakistan

The effect of plant age, the whitefly Bemisia tabaci population levels, and cultivar resistance to cotton leaf curl virus disease was determined for 10 newly developed mutant lines (via radiation) and one resistant and two susceptible/tolerant control varieties, under natural inoculation by the vector whiteflies. All cotton mutant lines/varieties become increasingly resistant to CLCuV as plants aged. Expression of the age-related resistance to CLCuV infection was more apparent in late growth stages. Maximum increase in % disease incidence occurred at 6 weeks. There was no significant correlation of whitefly levels with % disease incidence and only small differences in whitefly population levels for all mutant lines/varieties. None of the test lines/varieties under field or graft inoculation conditions were immune. All mutant lines including negative and positive controls were symptomatic but differed in their response to CLCuV. Six mutant lines, namely NIAB-103, NIAB-101, NIAB-998, NIAB-358, NIAB-98 and the negative control CIM-443 showed highly resistance responses while NIAB-78 and S-12 were highly susceptible. Similar results for disease reaction were obtained when test mutant lines were graft inoculated under net-house conditions (insect free conditions) except for CIM-443, which was resistant. Present results suggested that there is evidence that source of resistance against CLCuV is available in several of the tested mutant lines. These lines may be released as CLCuV resistant varieties or their resistance could be incorporated into other commercial cultivars to manage leaf curl disease. The current study furthered our knowledge about the epidemiology of CLCuV for improved forecasting to manage the disease and identified useful resistant materials.

Biochemical alterations in leaves of resistant and susceptible cotton genotypes infected systemically by cotton leaf curl Burewala virus

Leaf curl disease caused by Cotton Leaf Curl Burewala virus (CLCuBuV) has been recognized as serious threat to cotton in Indian subcontinent. However, information about cotton–CLCuBuV interaction is still limited. In this study, the level of phenolic compounds, total soluble proteins, and malondialdehyde (MDA) and the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POX), catalase (CAT), proteases, superoxide dismutase (SOD), and polyphenol oxidase (PPO) were studied in leaves of two susceptible (CIM-496 & NIAB-111) and two resistant (Ravi and Co Tiep Khac) cotton genotypes. Disease symptoms were mild in the resistant genotypes but were severe in highly susceptible genotypes. The results showed that phenolic compounds, proteins, PAL, POX, CAT, proteases, SOD, PPO, and MDA play an active role in disease resistance against CLCuBuV. The amount of total phenols, proteases, MDA, and PPO was significantly higher in leaves of CLCuBuV-inoculated plants of both resistant genotypes as in non-inoculated plants, and decreased in CLCuBuV-inoculated plants of both susceptible genotypes over their healthy plants. POX, protein content, SOD, and PAL activities showed lower values in resistant genotypes, while they decreased significantly in susceptible genotypes as compared to the noninoculated plants except PAL, which showed non-significant decrease. CAT was found to be increased in both susceptible and resistant genotypes with maximum percent increase in resistant genotype Ravi, as compared to non-inoculated plants. The results showed significantly higher concentrations of total phenols and higher activity of protease, MDA, SOD, and PPO in resistant genotype Ravi after infection with CLCuBuV, suggesting that there is a correlation between constitutive induced levels of these enzymes and plant resistance that could be considered as biochemical markers for studying plant-virus compatible and incompatible interactions.

An analysis of the resistance of Gossypium arboreum to cotton leaf curl disease by grafting

Cotton leaf curl disease (CLCuD) is a serious constraint to cotton production across Pakistan and north-western India. In the Punjab province of Pakistan at this time CLCuD is associated with one virus, Cotton leaf curl Burewala virus (CLCuBuV), and its associated betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB). Although some resistance to the virus complex causing CLCuD has been identified in Gossypium hirsutum, this has in the past proven not to be durable. At this time no commercial lines with strong resistance (immunity) to CLCuD are available. Although the cotton fibre produced by Gossypium arboreum is not of the quality required by processors, the species has a number of desirable characters, including resistance to a number of abiotic and biotic stresses, including CLCuD. For this reason there have been efforts to introgress CLCuD-resistance from G. arboreum into G. hirsutum. However, the nature of the resistance of G. arboreum to the viruses causing CLCuD remains unknown. An initial analysis of the resistance was herein conducted by graft inoculation. Graft inoculation of G. arboreum genotype Ravi with scions from severely infected G. hirsutum genotype CIM 496 plants showed that G. arboreum is able to support virus/betasatellite replication and long-distance spread. However, following grafting, only a small number of leaves developing at the time of grafting became mildly symptomatic and symptomatic tissue contained low amounts of virus/satellite DNA, as detected by Southern blot hybridisation; significantly less than the levels seen in G. hirsutum. The symptoms did not spread to the young upper leaves of graft inoculated plants, which contained DNA levels that could only be detected by polymerase chain reaction (PCR) or PCR following an initial amplification by rolling circle amplification–indicating very low DNA levels. The results showed that the resistance of G. arboreum to the virus complex causing CLCuD likely does not involve a mechanism that interferes with delivery of virus by the insect vector. Rather the resistance appears to be due to poor virus replication and possibly interference in short distance (cell-to-cell) spread.

Comparison of Resistance Level to Cotton leaf curl virus(CLCuV) Among Newly Developed Cotton Mutants and Commercial Cultivars

Four newly developed cotton mutants (M-111, M-7662, M-358 and M-218) were compared for their resistance against Cotton leaf curl virus(CLCuV) together with commercial resistant (CIM-443, CIM-482, CIM-473, FH-900 and FH-901) and susceptible (5-12) varieties by artificial inoculation through grafting and under natural field conditions. Infectivity and success of grafting were 100% in all cases. None of the grafted plants were found immune or asymptomatic. All the grafted mutants and most of their single plant progeny rows (SPPRs) showed highly resistant responses as the symptoms displayed by these mutants were milder than the commercial cultivars. Grafted mutants also had delayed disease reactions as they took more time (25-30 days) to produce disease symptoms, as compared with resistant commercial varieties that produced disease 18-22 days after inoculation. Growth of the grafted SPPRs of tested mutants was normal, which is an indication that there will be no production losses. Observations under natural infestation of whitefly showed that two SPPRs of M-ll/CE and M-7662-1/2 and one resistant variety CIM-443 exhibited slight incidence of disease, while one SPPR of M-l1/59 and S-12 were moderately susceptible and highly susceptible with 21％ and 97.l％ disease incidence, respectively. This study also showed that plants displaying more disease symptoms through grafting were easily infected under natural conditions. These results suggest that preference should be given to those plants that exhibited highly resistant responses after artificial inoculation.

CHINA ROSE (HIBISCUS ROSA-SINENSIS): A NEW NATURAL HOST OF COTTON LEAF CURL BUREWALA VIRUS IN PAKISTAN

China rose (Hibiscus rosa-sinensis) is a perennial ornamental plant grown throughout the tropics and subtropics. China rose plants with severe vein thickening/ greening, leaf curling, and enations on the lower leaf surface were found near cotton fields at the Nuclear Institute of Agriculture Biology (NIAB) in Faisalabad. The symptoms of disease exhibited on China rose plants were very much similar to Cotton leaf curl disease exhibited by cotton plants. DNA was extracted from ten naturally infected symptomatic China rose plants and subjected to PCR using begomovirus and betasatellite specific primers. The expected products of 2.8Kb and 1.4Kb for begomovirus and betasatellites were amplified, respectively. The begomovirus and its cognate betasatellite isolated from China rose were sequenced and submitted to the database with accession numbers HG003876 and HG003877, respectively. Owing to a 99% nucleotide sequence identity, this virus was designated as an isolate of Cotton leaf curl Burewala virus (CLCuBuV). Similarly the sequenced betasatellite showing 96% nucleotide sequence identity was designated as an isolate of Cotton leaf curl Multan betasatellite (CLCuMuB). The symptomatic induction of Cotton leaf curl disease (CLCuD) by indexing in LRA-5166, a CLCuBuV susceptible genotype of cotton that resists Cotton leaf curl Multan virus (CLCuMuV), further confirmed the presence of CLCuBuV in China rose. To our knowledge, this is the first report of CLCuBuV and its cognate betasatellite in infected China rose.

Occurrence of Cucumber mosaic virus Subgroup IA in tomato in Pakistan

Infection of tomato in Faisalabad, Pakistan with subgroup IA strain of Cucumber mosaic virus (CMV) is reported for the first time. The virus was detected by Rt-PCR using genus-specific single pair of primers and was transmitted by Myzus persicae in the non-persistent manner.ZusammenfassungMit der Untergruppe IA des Gurkenmosaikvirus infizierte Tomaten wurden erstmals in Pakistan nachgewiesen. Das Virus wurde mit einem einzelnen genusspezifischen Primer-paar mittels RT-PCR nachgewiesen. Die Übertragung durch Myzus persicae erfolgte nicht-persistent.

Sesbania bispinosa, a new host of a begomovirus-betasatellite complex in Pakistan

Abstract Symptoms of severe leaf curling, yellowing and vein thickening, typical of begomovirus infection, were observed on Sesbania bispinosa grown in cotton leaf curl disease affected cotton fields in Pakistan. A begomovirus and its associated betasatellite were amplified and sequenced from these affected plants. Complete nucleotide sequence showed that a monopartite begomovirus named Pedilanthus leaf curl virus (PeLCV) is associated with the disease and it shared highest sequence identity (98.73%) with a clone previously reported from soybean. Characterization of the betasatellite showed that it had highest sequence identity (96–97%) with Tobacco leaf curl betasatellite (TbLCB), previously isolated from soybean. Our data shows the expansion of the host range of begomovirus-betasatellite complexes. To the best of our knowledge, this is the first occurrence of PeLCV and TbLCB on S. bispinosa.

Transcriptomics reveals multiple resistance mechanisms against cotton leaf curl disease in a naturally immune cotton species, Gossypium arboreum

Cotton leaf curl disease (CLCuD), caused by cotton leaf curl viruses (CLCuVs), is among the most devastating diseases in cotton. While the widely cultivated cotton species Gossypium hirsutum is generally susceptible, the diploid species G. arboreum is a natural source for resistance against CLCuD. However, the influence of CLCuD on the G. arboreum transcriptome and the interaction of CLCuD with G. arboreum remains to be elucidated. Here we have used an RNA-Seq based study to analyze differential gene expression in G. arboreum under CLCuD infestation. G. arboreum plants were infested by graft inoculation using a CLCuD infected scion of G. hirsutum. CLCuD infested asymptomatic and symptomatic plants were analyzed with RNA-seq using an Illumina HiSeq. 2500. Data analysis revealed 1062 differentially expressed genes (DEGs) in G. arboreum. We selected 17 genes for qPCR to validate RNA-Seq data. We identified several genes involved in disease resistance and pathogen defense. Furthermore, a weighted gene co-expression network was constructed from the RNA-Seq dataset that indicated 50 hub genes, most of which are involved in transport processes and might have a role in the defense response of G. arboreum against CLCuD. This fundamental study will improve the understanding of virus-host interaction and identification of important genes involved in G. arboreum tolerance against CLCuD.

Evaluation of Bt-cotton Genotypes for Resistance to Cotton Leaf Curl Disease under High Inoculum Pressure in the Field and Using Graft Inoculation in Glasshouse.

Bt-cotton germplasm, consisting of 75 genotypes was evaluated against cotton leaf curl disease (CLCuD) under high inoculum pressure in the field and using graft inoculation in glasshouse by visual symptom scoring assessments. None of the tested genotype was found disease free under both evaluation tests. Under field conditions in 2011, 3 genotypes were found resistant, 4 moderately resistant, 3 tolerant, 2 moderately susceptible and one susceptible; in 2012, 3 genotypes were tolerant, 7 moderately susceptible, 5 susceptible and 38 highly susceptible; in 2013, one was moderately susceptible and 51 were highly susceptible with varying degree of percent disease index (PDI) and severity index (SI). However, through graft evaluation in glasshouse, none of the graft inoculated plant was symptomless. All tested genotypes showed disease symptoms with SI values ranging between 5.0 and 6.0, and latent period between 12 and 14 days. Of the 75 genotypes evaluated using graft inoculation, 11 were found susceptible with SI values of 5.0 to 5.4 while remaining 64 were highly susceptible with SI values of 5.5 to 6.0. Inoculated plants of all tested genotypes exhibited severe disease symptoms within 10 days after the appearance of initial symptoms. No reduction in SI value was observed until the end of the experiment i.e., 90 days after grafting. Information generated under the present study clearly demonstrates that no sources of resistance to CLCuD are available among the tested Bt-cotton genotypes. So, a breeding programme is needed to introgress the CLCuD-resistance from other resistant sources to agronomically suitable Bt-cotton genotypes.

PHYSIOLOGICAL RESPONSE OF COTTON LEAF CURL BUREWALA VIRUS-INFECTED PLANTS OF TOLERANT AND SUSCEPTIBLE GENOTYPES OF DIFFERENT GOSSYPIUM SPECIES

The effect of Cotton leaf curl Burewala virus (CLCuBuV) on cotton growth, yield, photosynthetic gas exchange, chlorophyll fluorescence and chlorophyll contents was studied in susceptible (CIM-496 and NIAB-111 from Gossypium hirsutum) and tolerant (Ravi from G. arboreum and Co Tiep Khac from G. herbaceum) cotton genotypes under glasshouse condition through artificial inoculation. Compared to the non-inoculated plants, the inoculated plants of susceptible genotypes showed stunted growth, reflected as significant decrease in plant height, internode length, number of leaves per plant, leaf area, number of bolls per plant, boll weight and yield per plant. Upon virus inoculation, transpiration rate decreased significantly in both susceptible genotypes while water use efficiency (WUE) was enhanced in CIM-496 compared to the non- inoculated plants. However, overall plant growth, net photosynthetic rate, leaf transpiration, stomatal conductance and WUE remained unaffected by CLCuBuV-infection in tolerant genotypes. Moreover, chlorophyll b and total chlorophyll content increased in one of the tolerant genotypes (Ravi) in response to CLCuBuV-infection while remaining unaffected in all other genotypes. Level of chlorophyll a and chlorophyll fluorescence (Fv/Fm) was not affected by CLCuBuV-infection in all genotypes. In conclusion, CLCuBuV infection adversely affected the overall plant growth and yield in susceptible genotypes while tolerant genotypes were able to maintain the growth and yield in the presence of infection. Reduced transpiration rate and increased WUE in susceptible genotypes and increased chlorophyll b and total chlorophyll content in tolerant genotypes on virus inoculation provided evidence for their significant involvement in CLCuBuV-tolerance mechanism in cotton.