First Report of Wilt Caused by Fusarium nanum (FIESC 25) on Mungbean (Vigna radiata) in Pakistan.

Mungbean, Vigna radia (L.) R. Wilczek, is ranked 2nd next to chickpea (Cicer arietinum) in total cultivation and production in Pakistan. In August of 2022 and 2023, mungbean plants (cv. PRI Mung-2018) were found wilting in a field at the Ayub Agricultural Research Institute, Faisalabad, Pakistan. Wilted leaves turned yellow, died, but remained attached to the stem. Vascular tissue at the base of the stem showed light to dark brown discoloration. Roots were stunted with purplish brown to black discoloration. Symptomatic mungbean plants were collected from fields at five different locations (20 samples/location). Disease incidence was similar among the five fields, ranging from 5 to 10% at each location depending upon type of germplasm and date of sowing. For fungal isolation and morphological identification, symptomatic stem and root tissues were cut into ~5 mm2 pieces with a sterilized blade. Tissues were surface-sterilized for one min in a 0.5% sodium hypochlorite solution, rinsed twice in sterilized water, air dried on sterilized filter paper, and aseptically placed on potato dextrose agar (PDA) containing 0.5 g/L-1 streptomycin sulphate. Plates were incubated for 3-4 days at 25 ± 2°C with a 12-h photoperiod. Single-spore cultures were used for morphological and molecular analyses. Isolates on PDA grew rapidly and produced abundant white aerial mycelium that turned off-white to beige with age. Macroconidia were hyaline, falcate, typically 3-to-6 septate with a pointed apical cell and a foot-shaped basal cell, measuring 24.5-49.5 x 2.7-4.7 µm (n = 40). Globose to obovate chlamydospores measuring 5.8 ± 0.5 µm (n = 40) were produced singly or in chains and were intercalary or terminal and possessed roughened walls. The morphological data indicated the isolates were members of the genus Fusarium (Leslie and Summerell 2006). To obtain a species-level identification, a portion of translation elongation factor 1-α (TEF1), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2) region were PCR amplified and sequenced using EF1/EF2 (O'Donnell et al. 1998), Fa/G2R (Hofstetter et al. 2007), and 5f2/7cr (Liu et al. 1999) primers, respectively. DNA sequences of these genes were deposited in GenBank under accession numbers MW059021, MW059017 and MW059019, respectively. The partial TEF1, RPB1 and RPB2 sequences were queried against the Fusarium MLST database (https://fusarium.mycobank.org/page/Fusarium_identification), using the polyphasic identification tool. The BLASTn search revealed 99.9% identity of the isolate to F. nanum (Xia et al. 2019), formerly FIESC 25 of the F. incarnatum-equiseti species complex (MRC 2610, NRRL 54143; O'Donnell et al. 2018). To confirm pathogenicity, roots of 3-5 leaf stage mungbean seedlings were soaked in a 106 spores ml-1 conidial suspension of the fungus for 15 min and then planted in 10 cm pots containing sterilized soil. Mock-inoculated plants with sterile water served as a negative control. Twenty pots that were used for each inoculated and control treatment were maintained at 25 ± 2°C, 14:8 h photoperiod, and 80% relative humidity in a growth chamber. After 15 days, leaf yellowing, internal browning from the base of stems and root discoloration was observed in all the inoculated plants. The uninoculated negative control plants remained asymptomatic. Fusarium nanum was re-isolated from artificially inoculated plants and identified by colony growth, conidial characteristics on PDA and molecular analyses (TEF1). To our knowledge, this is the first report of wilt caused by F.nanum on mungbean in Pakistan. In Pakistan, mungbean cultivation in irrigated areas has increased in recent years. It has been introduced frequently in citrus orchards, crop rotation of maize and sesame, intercropping with sugarcane and as green manure. However, citrus, maize, sesame and sugarcane are also hosts of Fusarium spp. Therefore, this information warrants sustainable crop protection and may have an impact on further interaction of F. nanum with other wilt pathogens.

SARS-CoV-2 nucleocapsid and Nsp3 binding: an in silico study.

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) belongs to the single-stranded positive-sense RNA family. The virus contains a large genome that encodes four structural proteins, small envelope (E), matrix (M), nucleocapsid phosphoprotein (N), spike (S), and 16 nonstructural proteins (nsp1-16) that together, ensure replication of the virus in the host cell. Among these proteins, the interactions of N and Nsp3 are essential that links the viral genome for processing. The N proteins reside at CoV RNA synthesis sites known as the replication-transcription complexes (RTCs). The N-terminal of N has RNA-binding domain (N-NTD), capturing the RNA genome while the C-terminal domain (N-CTD) anchors the viral Nsp3, a component of RTCs. Although the structural information has been recently released, the residues involved in contacts between N-CTD with Nsp3 are still unknown. To find the residues involved in interactions between two proteins, three-dimensional structures of both proteins were retrieved and docked using HADDOCK. Residues at N-CTD were detected in interaction with L499, R500, K501, V502, P503, T504, D505, N506, Y507, I508, T509, K529, K530K532, S533 of Nsp3 and N-NTD to synthesize SARS-CoV-2 RNA. The interaction between Nsp3 and CTD of N protein may be a potential drug target. The current study provides information for better understanding the interaction between N protein and Nsp3 that could be a possible target for future inhibitors.

A comprehensive review on Gossypium hirsutum resistance against cotton leaf curl virus.

Cotton (Gossypium hirsutum L.) is a significant fiber crop. Being a major contributor to the textile industry requires continuous care and attention. Cotton is subjected to various biotic and abiotic constraints. Among these, biotic factors including cotton leaf curl virus (CLCuV) are dominant. CLCuV is a notorious disease of cotton and is acquired, carried, and transmitted by the whitefly (Bemisia tabaci). A cotton plant affected with CLCuV may show a wide range of symptoms such as yellowing of leaves, thickening of veins, upward or downward curling, formation of enations, and stunted growth. Though there are many efforts to protect the crop from CLCuV, long-term results are not yet obtained as CLCuV strains are capable of mutating and overcoming plant resistance. However, systemic-induced resistance using a gene-based approach remained effective until new virulent strains of CLCuV (like Cotton Leaf Curl Burewala Virus and others) came into existence. Disease control by biological means and the development of CLCuV-resistant cotton varieties are in progress. In this review, we first discussed in detail the evolution of cotton and CLCuV strains, the transmission mechanism of CLCuV, the genetic architecture of CLCuV vectors, and the use of pathogen and nonpathogen-based approaches to control CLCuD. Next, we delineate the uses of cutting-edge technologies like genome editing (with a special focus on CRISPR-Cas), next-generation technologies, and their application in cotton genomics and speed breeding to develop CLCuD resistant cotton germplasm in a short time. Finally, we delve into the current obstacles related to cotton genome editing and explore forthcoming pathways for enhancing precision in genome editing through the utilization of advanced genome editing technologies. These endeavors aim to enhance cotton's resilience against CLCuD.

Detection of quantitative trait loci for mungbean yellow mosaic India virus (MYMIV) resistance in mungbean (Vigna radiata (L.) Wilczek) in India and Pakistan.

Yellow mosaic disease (YMD) is one of the major diseases affecting mungbean (Vigna radiata (L.) Wilczek). In this study, we report the mapping of the quantitative trait locus (QTL) for mungbean yellow mosaic India virus (MYMIV) resistance in mungbean. An F8 recombinant inbred line (RIL) mapping population was generated in Thailand from a cross between NM10-12-1 (MYMIV resistance) and KPS2 (MYMIV susceptible). One hundred and twenty-two RILs and their parents were evaluated for MYMIV resistance in infested fields in India and Pakistan. A genetic linkage map was developed for the RIL population using simple sequence repeat (SSR) markers. Composite interval mapping identified five QTLs for MYMIV resistance: three QTLs for India (qYMIV1, qYMIV2 and qYMIV3) and two QTLs for Pakistan (qYMIV4 and qYMIV5). qYMIV1, qYMIV2, qYMIV3, qYMIV4 and qYMIV5 explained 9.33%, 10.61%, 12.55%, 21.93% and 6.24% of variation in disease responses, respectively. qYMIV1 and qYMIV4 appeared to be the same locus and were common to a major QTL for MYMIV resistance in India identified previously using a different resistant mungbean.

Friction and Wear Performance Evaluation of Bio-Lubricants and DLC Coatings on Cam/Tappet Interface of Internal Combustion Engines.

The environmental concerns associated with artificially formulated engine oils have forced a shift towards bio-based lubricants. The deposition of hard coatings on engine components and migrating to environmentally friendly green lubricants can help in this regard. Chemically modified forms of vegetable oils, with better low-temperature characteristics and enhanced thermo-oxidative stability, are suitable substitutes to conventional lubricant base oils. The research presented in this manuscript was undertaken to experimentally investigate the wear and friction performance of a possible future generation of an environmentally friendly bio-based lubricant as a potential replacement for conventional engine lubricants. In order to quantify the tribological benefits which can be gained by the deposition of DLC coatings, (an (a-C:H) hydrogenated DLC coating and an (a-C:H:W) tungsten-doped DLC coating) were applied on the cam/tappet interface of a direct acting valve train assembly of an internal combustion engine. The tribological correlation between DLC-coated engine components, lubricant base oils and lubricant additives have been thoroughly investigated in this study using actual engine operating conditions. Two additive-free base oils (polyalphaolefines (PAO) and chemically-modified palm oil (TMP)) and two multi-additive-containing lubricants were used in this investigation. Real-time drive torque was measured to determine the friction force, detailed post-test analysis was performed, which involved the use of a specialized jig to measure camlobe wear. An optical profilometer was used to measure the wear on the tappet, high-resolution scanning electron microscopy was employed to study the wear mechanism and energy-dispersive X-ray spectroscopy was performed on the tested samples to qualitatively access the degradation of the coating. When using additive-free TMP, a low friction coefficient was observed for the cam/tappet interface. The presence of additives further improved the friction characteristics of TMP, resulting in reduced average friction torque values. A tremendous enhancement in wear performance was recorded with a-C:H-coated parts and the coating was able to withstand the test conditions with little or no delamination.

Pyrazinamide resistance of novel mutations in pncA and their dynamic behavior.

Pyrazinamide (PZA) is one of the essential anti-mycobacterium drugs, active against non-replicating Mycobacterium tuberculosis (MTB) isolates. PZA is converted into its active state, called pyrazinoic acid (POA), by action of pncA encoding pyrazinamidase (PZase). In the majority of PZA-resistance isolates, pncA harbored mutations in the coding region. In our recent report, we detected a number of novel variants in PZA-resistance (PZAR) MTB isolates, whose resistance mechanisms were yet to be determined. Here we performed several analyses to unveil the PZAR mechanism of R123P, T76P, G150A, and H71R mutants (MTs) through molecular dynamics (MD) simulations. In brief, culture positive MTB isolates were subjected to PZA susceptibility tests using the WHO recommended concentration of PZA (100 µg ml-1). The PZAR samples were screened for mutations in pncA along sensitive isolates through polymerase chain reactions and sequencing. A large number of variants (GeneBank accession no. MH461111), including R123P, T76P, G150A, and H71R, have been spotted in more than 70% of isolates. However, the mechanism of PZAR for mutants (MTs) R123P, T76P, G150A, and H71R was unknown. For the MTs and native PZase structures (WT), thermodynamic properties were compared using molecular dynamics simulations for 100 ns. The MTs structural activity was compared to the WT. Folding effect and pocket volume variations have been detected when comparing between WT and MTs. Geometric matching further confirmed the effect of R123P, T76P, G150A, and H71R mutations on PZase dynamics, making them vulnerable for activating the pro-drug into POA. This study offers a better understanding for management of PZAR TB. The results may be used as alternative diagnostic tools to infer PZA resistance at a structural dynamics level.

Structural and free energy landscape of novel mutations in ribosomal protein S1 (rpsA) associated with pyrazinamide resistance.

Resistance to key first-line drugs is a major hurdle to achieve the global end tuberculosis (TB) targets. A prodrug, pyrazinamide (PZA) is the only drug, effective in latent TB, recommended in drug resistance and susceptible Mycobacterium tuberculosis (MTB) isolates. The prodrug conversion into active form, pyrazinoic acid (POA), required the activity of pncA gene encoded pyrazinamidase (PZase). Although pncA mutations have been commonly associated with PZA resistance but a small number of resistance cases have been associated with mutationss in RpsA protein. Here in this study a total of 69 PZA resistance isolates have been sequenced for pncA mutations. However, samples that were found PZA resistant but pncA wild type (pncAWT), have been sequenced for rpsA and panD genes mutation. We repeated a drug susceptibility testing according to the WHO guidelines on 18 pncAWT MTB isolates. The rpsA and panD genes were sequenced. Out of total 69 PZA resistant isolates, 51 harbored 36 mutations in pncA gene (GeneBank Accession No. MH46111) while, fifteen different mutations including seven novel, were detected in the fourth S1 domain of RpsA known as C-terminal (MtRpsACTD) end. We did not detect any mutations in panD gene. Among the rpsA mutations, we investigated the molecular mechanism of resistance behind mutations, D342N, D343N, A344P, and I351F, present in the MtRpsACTD through molecular dynamic simulations (MD). WT showed a good drug binding affinity as compared to mutants (MTs), D342N, D343N, A344P, and I351F. Binding pocket volume, stability, and fluctuations have been altered whereas the total energy, protein folding, and geometric shape analysis further explored a significant variation between WT and MTs. In conclusion, mutations in MtRpsACTD might be involved to alter the RpsA activity, resulting in drug resistance. Such molecular mechanism behind resistance may provide a better insight into the resistance mechanism to achieve the global TB control targets.

Susceptibility of cotton whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) to diverse pesticides in Pakistan.

The cotton whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a serious pest of several summer crops in hot and dry climates. Its field populations (Asia II-1 biotype) were assessed for their susceptibility to diverse pesticides by using leaf-dip bioassay. There was no or a very low resistance to amitraz, hexythiazox, and pyridaben during 1992-2015. B. tabaci also exhibited no resistance to endosulfan during 1992-1997 and a very low resistance during 1998-2010, which then rose to a low level during 2011-2015. Chlorfenapyr resistance was very low during 1997-2008 and it reached to a high level during 2009-2011 and to a very high level in 2013 and 2015. Among avermectins, abamectin showed a very low resistance up to 2013 but a high resistance in 2015. Emamectin benzoate also demonstrated a very low resistance up to 2010, but a moderate-to-high resistance during 2011-2015. It may be concluded that the diverse chemistries, having novel modes of action and showing no, very low or low levels of resistance, can be substituted in rotation in the wake of resistance development to conventional insecticides.

Molecular Confirmation of Intraspecific Tomato (Solanum lycopersicum) Hybrids and Their Evaluation Against Late Blight and Cucumber Mosaic Virus.

Tomato is one of the most consumed vegetables in the world. Diseases are the number one concern in the development of high-yield and disease-resistant tomato hybrids which is the foremost priority of breeders. Present study was conducted (1) to develop DNA-based markers for genetic confirmation of tomato F1 hybrids, (2) to utilize sequenced characterized amplified region (SCAR) marker linked to the Ph-3 gene for Phytophthora infestans resistance in tomato and (3) to evaluate male and female parental genotypes and their F1 hybrids against late blight (LB) and cucumber mosaic virus (CMV). For molecular studies, 58 previously reported markers including RAPDs (10), SCAR (01), EST-SSR (01) and SSR (46) were applied. The SCAR marker clearly differentiated the LB3 and LB4 from Roma and T-1359 and provided evidence for Ph-3 gene. The SCAR marker was able to confirm the Ph-3 gene in the hybrids Roma × LB4, Roma × LB3, Riogrande × LB2, Riogrande × LB3 and Roma × LB7. Out of several tested primers, SSR-22 proved useful for genetic confirmation of F1 hybrid TMS1 × Money Maker (MM). For LB, tested hybrids/genotypes were ranked as susceptible to highly susceptible with different infection percentage (IP). However, the pace of symptom development was slower in hybrid Rio × LB2, 45% IP after 10 days of inoculation compared with 85% disease in one of the parent genotypes (Riogrande). None of the tested genotypes was found resistant; however, TMS1 responded as tolerant against CMV using mechanical inoculation. Under natural field conditions, TMS1 was found resistant while hybrids TMS1 × Naqeeb and TMS1 × MM were tolerant where as others were found to be susceptible. In conclusion, all tomato hybrids were genetically confirmed using DNA-based markers. SCAR marker was useful for marker-assisted confirmation of the Ph-3 gene in parental lines and hybrids; however, this gene was unable to provide protection against the local population of P. infestans.

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.

Diversity in Betasatellites Associated with Cotton Leaf Curl Disease During Source-To-Sink Movement Through a Resistant Host.

Cotton leaf curl is devastating disease of cotton characterized by leaf curling, vein darkening and enations. The disease symptoms are induced by DNA satellite known as Cotton leaf curl Multan betasatellite (CLCuMuB), dominant betasatellite in cotton but another betasatellite known as Chili leaf curl betasatellite (ChLCB) is also found associated with the disease. Grafting experiment was performed to determine if host plant resistance is determinant of dominant population of betasatellite in cotton (several distinct strains of CLCuMuB are associated with the disease). Infected scion of Gossypium hirsutum collected from field (the source) was grafted on G. arboreum, a diploid cotton species, resistant to the disease. A healthy scion of G. hirsutum (sink) was grafted at the top of G. arboreum to determine the movement of virus/betasatellite to upper susceptible scion of G. hirsutum. Symptoms of disease appeared in the upper scion and presence of virus/betasatellite in the upper scion was confirmed via molecular techniques, showing that virus/betasatellite was able to move to upper scion through resistant G. arboreum. However, no symptoms appeared on G. arboreum. Betasatelites were cloned and sequenced from lower scion, upper scion and G. arboreum which show that the lower scion contained both CLCuMuB and ChLCB, however only ChLCB was found in G. arboreum. The upper scion contained CLCuMuB with a deletion of 78 nucleotides (nt) in the non-coding region between A-rich sequence and ßC1 gene and insertion of 27 nt in the middle of ßC1 ORF. This study may help in investigating molecular basis of resistance in G. arboreum.

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.