Phylogenetic and recombination analysis of Begomoviruses associated with Cotton leaf curl disease and in silico analysis of viral-host protein interactions.

Cotton leaf curl disease (CLCuD), caused by numerous begomoviruses (BGVs), is a highly disastrous disease in cotton crops worldwide. To date, several efforts have shown limited success in controlling this disease. CLCuD-associated BGVs (CABs) are known for their high rate of intra and interspecific recombinations, which raises an urgent need to find an efficient and conserved target region to combat disease. In the present study, phylogenetic analysis of selected 11 CABs, along with associated alphasatellites, and betasatellites revealed a close evolutionary relationship among them. Recombination analysis of 1374 isolates of CABs revealed 54 recombination events for the major players of CLCuD in cotton and the Cotton leaf curl Multan virus (CLCuMuV) as the most recombinant CAB. Recombination breakpoints were frequent in all regions except C2 and C3. C3-encoded protein, known as viral replication enhancer (REn), promotes viral replication by enhancing the activity of replicase (Rep) protein. Both proteins were found to contain significantly conserved domains and motifs. The identified motifs were found crucial for their interaction with host protein PCNA (Proliferating cell nuclear antigen), facilitating viral replication. Interruption at the REn-PCNA and Rep-PCNA interactions by targeting the identified conserved motifs is proposed as a prospect to halt viral replication, after suitable experimental validation.

Development, Design, and Application of Efficient siRNAs Against Cotton Leaf Curl Virus-Betasatellite Complex to Mediate Resistance Against Cotton Leaf Curl Disease.

Cotton leaf curl disease (CLCuD), caused by the Cotton leaf curl virus, is one of the most irrepressible diseases in cotton due to high recombination in the virus. RNA interference (RNAi) is widely used as a biotechnological approach for sequence-specific gene silencing guided by small interfering RNAs (siRNAs) to generate resistance against viruses. The success of RNAi depends upon the fact that the target site of the designed siRNA must be conserved even if the genome undergoes recombination. Thus, the present study designs the most efficient siRNA against the conserved sites of the Cotton leaf curl Multan virus (CLCuMuV) and the Cotton leaf curl Multan betasatellite (CLCuMB). From an initial prediction of 9 and 7 siRNAs against CLCuMuV and CLCuMB, respectively, the final selection was made for 2 and 1 siRNA based on parameters such as no off-targets, good GC content, high validity score, and targeting coding region. The target sites of siRNA were observed to lie in the AC3 and an overlapping region of AC2-AC1 of CLCuMuV and ßC1 of CLCuMB; all target sites showed a highly conserved nature in recombination analysis. Docking the designed siRNAs with the Argonaute-2 protein of Gossypium hirsutum showed stable binding. Finally, BLASTn of siRNA-target positions in genomes of other BGVs indicated the suitability of designed siRNAs against a broad range of BGVs. The designed siRNAs of the present study could help gain complete control over the virus, though experimental validation is highly required to suggest predicted siRNAs for CLCuD resistance. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01191-z.

Low Temperature Inhibits the Defoliation Efficiency of Thidiazuron in Cotton by Regulating Plant Hormone Synthesis and the Signaling Pathway.

Thidiazuron (TDZ) is the main defoliant used in production to promote leaf abscission for machine-picked cotton. Under low temperatures, the defoliation rate of cotton treated with TDZ decreases and the time of defoliation is delayed, but there is little information about this mechanism. In this study, RNA-seq and physiological analysis are performed to reveal the transcriptome profiling and change in endogenous phytohormones upon TDZ treatment in abscission zones (AZs) under different temperatures (daily mean temperatures: 25 °C and 15 °C). Genes differentially expressed in AZs between TDZ treatment and control under different temperatures were subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses to compare the enriched GO terms and KEGG pathways between the two temperature conditions. The results show that, compared with the corresponding control group, TDZ induces many differentially expressed genes (DEGs) in AZs, and the results of the GO and KEGG analyses show that the plant hormone signaling transduction pathway is significantly regulated by TDZ. However, under low temperature, TDZ induced less DEGs, and the enriched GO terms and KEGG pathways were different with those under normal temperature condition. Many genes in the plant hormone signal transduction pathway could not be induced by TDZ under low temperature conditions. In particular, the upregulated ethylene-signaling genes and downregulated auxin-signaling genes in AZs treated with TDZ were significantly affected by low temperatures. Furthermore, the expression of ethylene and auxin synthesis genes and their content in AZs treated with TDZ were also regulated by low temperature conditions. The upregulated cell wall hydrolase genes induced by TDZ were inhibited by low temperatures. However, the inhibition of low temperature on genes in AZs treated with TDZ was relieved with the extension of the treatment time. Together, these results indicate that the responses of ethylene and auxin synthesis and the signaling pathway to TDZ are inhibited by low temperatures, which could not induce the expression of cell wall hydrolase genes, and then inhibit the separation of AZ cells and the abscission of cotton leaves. This result provides new insights into the mechanism of defoliation induced by TDZ under low temperature conditions.

Morpho-physiological and biochemical responses of cotton (Gossypium hirsutum L.) genotypes upon sucking insect-pest infestations.

The effects of sucking insect-pests on the morpho-physiological and biochemical changes in the leaves of four cotton genotypes-Bio 100 BG-II and GCH-3 (highly tolerant); KDCHH-9810 BG-II and HS-6 (highly susceptible)-were examined. Compared to tolerant genotypes, susceptible genotypes showed a decrease in relative water content, specific leaf weight, leaf area, photosynthetic rate, and total chlorophyll content, with an increase in electrolyte leakage. Hydrogen peroxide and total soluble sugar content were higher in susceptible plants. In contrast, resistant plants had higher levels of total soluble protein, total phenolic content, gossypol content, tannin content, peroxidase activity, and polyphenol oxidase. The findings demonstrated that the Bio 100 BG-II and GCH-3 genotypes effectively offset the impact of sucking insect-pests by modifying the factors mentioned above. The KDCHH-9810 BG-II and HS-6 genotypes could not completely negate the effects of sucking insect-pests. Customized metabolites and total soluble protein are more efficient in protecting cotton plants from damage brought on by infestations of sucking insects and pests. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01253-w.

Occurrence of Cotton leaf curl Multan virus and associated betasatellites with leaf curl disease of Bhut-Jolokia chillies (Capsicum chinense Jacq.) in India.

Geminiviridae comprises the largest family of plant viruses which causes severe crop losses in India. The highest pungency chilli Bhut-Jolokia or ghost pepper (Capsicum chinense Jaqc.) hails from North-East region of India and is used in many dishes to add flavors and also for its medicinal value. However, this chilli variety is also affected by viruses leading to crop and economic losses. The present study reports the identification of begomoviruses in the infected chilli Bhut-Jolokia leaf samples collected from eight different places of North-East region (Manipur) of India. The infected leaf samples were screened for the presence of viral genome by rolling circle amplification (RCA) followed by PCR using degenerate primer pairs. The subsequent analyses using restriction fragment length polymorphism and sequencing revealed the presence of Cotton leaf curl Multan virus (CLCuMuV), and Tomato leaf curl Patna betasatellite (ToLCPaB). The findings focus on the phylogenetic relatedness, probable recombinational hot-spots and evolutionary divergence of the viral DNA sequences with the current reported begomoviral genome. To the best of our knowledge, this is the first report showing the presence of CLCuMuV, and associated non-cognate ToLCPaB with leaf curl disease of Bhut-Jolokia chillies. The study reveals potential recombination sites on both viral genome and betsatellite which, during the course of evolution, may have aided the virus to progress and successfully establish infection in chilli plants. Taken together, our results suggest a possible spread of CLCuMuV to the hitherto non-host crop in the North-East region of India.

Diversity and recombination analysis of Cotton leaf curl Multan virus: a highly emerging begomovirus in northern India.

BACKGROUND: Cotton leaf curl disease (CLCuD), caused by begomoviruses in association with satellite molecules, is a major threat to cotton production causing enormous losses to cotton crop in most of the cotton growing countries including Indian subcontinent. In this study, isolates of begomovirus and satellite molecules associated with CLCuD were collected from North India (Haryana, New Delhi). They were amplified employing rolling circle replication mechanism, cloned, sequenced and, their phylogenetic and recombination analysis was performed. RESULTS: The five Cotton leaf curl Multan virus (CLCuMuV) isolates investigated in this study showed monopartite organization of the genome typical of Old World begomoviruses. Nucleotide sequence analyses assigned them as the strains of CLCuMuV and were designated as CLCuMuV-SR13, CLCuMuV-SR14, CLCuMuV-ND14, CLCuMuV-ND15 and CLCuMuV-SR15. The genome of CLCuMuV-SR13 shared a highest level of nucleotide sequence identity (98%) with CLCuMuV (JN678804), CLCuMuV-SR14 and CLCuMuV-SR15 exhibited 96% with CLCuMuV (KM096471), while isolates CLCuMuV-ND15 and CLCuMuV-SR15 revealed 96% sequence identity with CLCuMuV (AY765253). The four betasatellite molecules investigated in this study shared 95-99% nucleotide sequence identity with Cotton leaf curl Multan betasatellite (CLCuMB) from India. The betasatellite molecules were designated as CLCuMB-SR13, CLCuMB-SR14, CLCuMB-ND14 and CLCuMB-ND15. Alphasatellite molecules in this study, designated as GLCuA-SR14, GLCuA-ND14 and GLCuA-SR15, revealed 98% identity with Guar leaf curl alphasatellite (GLCuA) reported from Pakistan. CONCLUSION: The phylogenetic and recombination studies concluded that the isolates of CLCuMuV genomes undertaken in this study have a potential recombinant origin. Remarkably, significant recombination was detected in almost all the genes with contribution of Cotton leaf curl Kokhran Virus (CLCuKoV) in IR, V1, V2, C1, C4 and C5 regions and of CLCuMuV in C2 region of CLCuMuV-SR14. CLCuKoV also donated in C2, C3 regions of CLCuMuV-ND14; V1, V2, C2 and C3 regions of CLCuMuV-ND15 and C1 of CLCuMuV-SR15. Altogether, these observations signify the uniqueness in Indian CLCuMuV isolates showing contribution of CLCuKoV in all the genes. An interesting observation was frequent identification of GLCuA in CLCuD leaf samples.

An improved protein extraction method applied to cotton leaves is compatible with 2-DE and LC-MS.

BACKGROUND: Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used in plant proteomics research. However, these two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome. RESULTS: Here, we evaluated the efficiency of three different protein extraction methods for 2-DE and LC-MS/MS analyses of total proteins obtained from cotton leaves. The protein yield of the borax/PVPP/phenol (BPP) method (0.14%) was significantly lower than the yields of the trichloroacetic acid/acetone (TCA) precipitation method (1.42%) and optimized TCA combined with BPP (TCA-B) method (0.47%). The BPP method was failed to get a clear 2-DE electrophoretogram. Fifty pairs of protein spots were randomly selected from the 2-DE gels of TCA- and TCA-B-extracted proteins for identification by MALDI TOF/TOF, and the results of 42 pairs were consistent. High-throughput proteomic analysis showed that 6339, 9282 and 9697 unique proteins were identified from the total cotton leaf proteins extracted by the TCA, BPP and TCA-B methods, respectively. Gene Ontology (GO) analysis revealed that the proteins specifically identified by TCA method were primarily distributed in the plasma membrane, while BPP and TCA-B methods specific proteins distributed in the cytosol, indicating the sub-cellular preference of different protein extraction methods. Further, ATP-dependent zinc metalloprotease FTSH 8 could be observed in the 2-DE gels of TCA and TCA-B methods, and could only be detected in the LC-MS/MS results of the BPP and TCA-B methods, showing that TCA-B method might be the optimized choice for both 2-DE and LC-MS/MS. CONCLUSION: Our data provided an improved TCA-B method for protein extraction that is compatible with 2-DE and LC-MS/MS for cotton leaves and similar plant tissues which is rich in polysaccharides and polyphenols.

RNAi-mediated resistance against Cotton leaf curl disease in elite Indian cotton (Gossypium hirsutum) cultivar Narasimha.

Cotton leaf curl disease (CLCuD) is caused by several distinct begomovirus species in association with disease-specific betasatellite essential for induction of disease symptoms. CLCuD is a serious threat for the cultivation of cotton (Gossypium sp.) and several species in the family Malvaceae. In this study, RNAi-based approach was applied to generate transgenic cotton (Gossypium hirsutum) plants resistant to Cotton leaf curl Rajasthan virus (CLCuRV). An intron hairpin (ihp) RNAi construct capable of expressing dsRNA homologous to the intergenic region (IR) of CLCuRV was designed and developed. Following Agrobacterium tumefaciens-mediated transformation of cotton (G. hirsutum cv. Narasimha) plants with the designed ihpRNAi construct, a total of 9 independent lines of transformed cotton were obtained. The presence of the potential stretch of IR in the transformed cotton was confirmed by PCR coupled with Southern hybridization. Upon inoculation with viruliferous whiteflies, the transgenic plants showed high degree of resistance. None of them displayed any CLCuD symptoms even after 90 days post inoculation. The transformed cotton plants showed the presence of siRNAs. The present study demonstrated that ihp dsRNA-mediated resistance strategy of RNAi is an effective means to combat the CLCuD infection in cotton.

Identification and characterization of a fatty acyl reductase from a Spodoptera littoralis female gland involved in pheromone biosynthesis.

Fatty acyl-CoA reductases (FARs), the enzymes that catalyse reduction of a fatty acyl-CoA to the corresponding alcohol in insect pheromone biosynthesis, are postulated to play an important role in determining the proportion of each component in the pheromone blend. For the first time, we have isolated and characterized from the Egyptian cotton leaf worm Spodoptera littoralis (Lepidoptera: Noctuidae) a FAR cDNA (Slit-FAR1), which appeared to be expressed only in the pheromone gland and was undetectable in other female tissues, such as fat body, ovaries, wings, legs or thorax. The encoded protein has been successfully expressed in a recombinant system, and the recombinant enzyme is able to produce the intermediate fatty acid alcohols of the pheromone biosynthesis of S. littoralis from the corresponding acyl-CoA precursors. The kinetic variables Km and Vmax, which have been calculated for each acyl-CoA pheromone precursor, suggest that in S. littoralis pheromone biosynthesis other biosynthetic enzymes (e.g. desaturases, acetyl transferase) should also contribute to the final ratio of components of the pheromone blend. In a phylogenetic analysis, Slit-FAR1 appeared grouped in a cluster of other FARs involved in the pheromone biosynthesis of other insects, with little or non-specificity for the natural pheromone precursors.

Comparative proteomics of Bt-transgenic and non-transgenic cotton leaves.

BACKGROUND: As the rapid growth of the commercialized acreage in genetically modified (GM) crops, the unintended effects of GM crops' biosafety assessment have been given much attention. To investigate whether transgenic events cause unintended effects, comparative proteomics of cotton leaves between the commercial transgenic Bt + CpTI cotton SGK321 (BT) clone and its non-transgenic parental counterpart SY321 wild type (WT) was performed. RESULTS: Using enzyme linked immunosorbent assay (ELISA), Cry1Ac toxin protein was detected in the BT leaves, while its content was only 0.31 pg/g. By 2-DE, 58 differentially expressed proteins (DEPs) were detected. Among them 35 were identified by MS. These identified DEPs were mainly involved in carbohydrate transport and metabolism, chaperones related to post-translational modification and energy production. Pathway analysis revealed that most of the DEPs were implicated in carbon fixation and photosynthesis, glyoxylate and dicarboxylate metabolism, and oxidative pentose phosphate pathway. Thirteen identified proteins were involved in protein-protein interaction. The protein interactions were mainly involved in photosynthesis and energy metabolite pathway. CONCLUSIONS: Our study demonstrated that exogenous DNA in a host cotton genome can affect the plant growth and photosynthesis. Although some unintended variations of proteins were found between BT and WT cotton, no toxic proteins or allergens were detected. This study verified genetically modified operation did not sharply alter cotton leaf proteome, and the target proteins were hardly checked by traditional proteomic analysis.

Temporal changes in the levels of virus and betasatellite DNA in B. tabaci feeding on CLCuD affected cotton during the growing season.

Cotton, a key source of income for Pakistan, has suffered significantly by cotton leaf curl disease (CLCuD) since 1990. This disease is caused by a complex of phylogenetically-related begomovirus (genus Begomovirus, family Geminiviridae) species and a specific betasatellite (genus Betasatellite, family Tolecusatellitidae), cotton leaf curl Multan betasatellite. Additionally, another DNA satellite called alphasatellite (family Alphasatellitidae), is also frequently associated. All these virus components are vectored by a single species of whitefly (Bemisia tabaci). While many factors affect cotton productivity, including cotton variety, sowing time, and environmental cues such as temperature, humidity, and rainfall, CLCuD is a major biotic constraint. Although the understanding of begomoviruses transmission by whiteflies has advanced significantly over the past three decades, however, the in-field seasonal dynamics of the viruses in the insect vector remained an enigma. This study aimed to assess the levels of virus and betasatellite in whiteflies collected from cotton plants throughout the cotton growing season from 2014 to 2016. Notably, begomovirus levels showed no consistent pattern, with minimal variations, ranging from 0.0017 to 0.0074 ng.µg-1 of the genomic DNA in 2014, 0.0356 to 0.113 ng.µg-1 of the genomic DNA in 2015, and 0.0517 to 0.0791 ng.µg-1 of the genomic DNA in 2016. However, betasatellite levels exhibited a distinct pattern. During 2014 and 2015, it steadily increased throughout the sampling period (May to September). While 2016 showed a similar trend from the start of sampling (July) to September but a decline in October (end of sampling). Such a study has not been conducted previously, and could potentially provide valuable insights about the epidemiology of the virus complex causing CLCuD and possible means of controlling losses due to it.

Gossypium hirsutum calmodulin-like protein (CML 11) interaction with geminivirus encoded protein using bioinformatics and molecular techniques.

Plant viruses are the most abundant destructive agents that exist in every ecosystem, causing severe diseases in multiple crops worldwide. Currently, a major gap is present in computational biology determining plant viruses interaction with its host. We lay out a strategy to extract virus-host protein interactions using various protein binding and interface methods for Geminiviridae, a second largest virus family. Using this approach, transcriptional activator protein (TrAP/C2) encoded by Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan virus (CLCuMV) showed strong binding affinity with calmodulin-like (CML) protein of Gossypium hirsutum (Gh-CML11). Higher negative value for the change in Gibbs free energy between TrAP and Gh-CML11 indicated strong binding affinity. Consensus from gene ontology database and in-silico nuclear localization signal (NLS) tools identified subcellular localization of TrAP in the nucleus associated with Gh-CML11 for virus infection. Data based on interaction prediction and docking methods present evidences that full length and truncated C2 strongly binds with Gh-CML11. This computational data was further validated with molecular results collected from yeast two-hybrid, bimolecular fluorescence complementation system and pull down assay. In this work, we also show the outcomes of full length and truncated TrAP on plant machinery. This is a first extensive report to delineate a role of CML protein from cotton with begomoviruses encoded transcription activator protein.

Comparative transcriptome analysis of whiteflies raised on cotton leaf curl Multan virus-infected cotton plants.

Cotton leaf curl Multan virus (CLCuMuV), a serious viral disease causative agent in cotton plants in South Asia, is transmitted by the Bemisia tabaci cryptic species complex in a persistent circulative manner. A previous study indicated that Asia II-7 whiteflies could transmit CLCuMuV, while Mediterranean (MED) whiteflies failed to transmit CLCuMuV. However, little is known about the genes involved in this process. In this study, Asia II-7 and MED B. tabaci were utilized to determine transcriptomic responses after 48 h of acquisition access periods (AAPs). Result of Illumina sequencing revealed that, 14,213 and 8,986 differentially expressed genes (DEGs) were identified. Furthermore, DEGs related to the immune system and metabolism of Asia II-7 and MED in response to CLCuMuV-infected plants were identified and analyzed using Gene Ontologies (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the number of related DEGs in MED was lower than that of Asia II-7. The most abundant groups of DEGs between both viruliferous and aviruliferous whitefly species were the zf-C2H2 family of transcription factors (TFs). Notably, in comparison to viruliferous MED, Asia II-7 exhibited more DEGs related to cathepsin biosynthesis. Overall, this study provides the basic information for investigating the molecular mechanism of how begomoviruses affect B. tabaci metabolism and immune response either as vector cryptic species or non-vector species.

Dominance of Cotton leaf curl Multan virus-Rajasthan strain associated with third epidemic of cotton leaf curl disease in Pakistan.

Cotton (Gossypium hirsutum) is an economically potent crop in many countries including Pakistan, India, and China. For the last three decades, cotton production is under the constant stress of cotton leaf curl disease (CLCuD) caused by begomoviruses/satellites complex that is transmitted through the insect pest, whitefly (Bemisia tabaci). In 2018, we identified a highly recombinant strain; Cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Raj), associated with the Cotton leaf curl Multan betasatellite-Vehari (CLCuMuBVeh). This strain is dominant in cotton-growing hub areas of central Punjab, Pakistan, causing the third epidemic of CLCuD. In the present study, we have explored the CLCuD diversity from central to southern districts of Punjab (Faisalabad, Lodhran, Bahawalpur, Rahimyar Khan) and the major cotton-growing region of Sindh (Tandojam), Pakistan for 2 years (2020-2021). Interestingly, we found same virus (CLCuMuV-Raj) and associated betasatellite (CLCuMuBVeh) strain that was previously reported with the third epidemic in the central Punjab region. Furthermore, we found minor mutations in two genes of CLCuMuV-Raj C4 and C1 in 2020 and 2021 respectively as compared to its isolates in 2018, which exhibited virus evolution. Surprisingly, we did not find these mutations in CLCuMuV-Raj isolates identified from Sindh province. The findings of the current study represent the stability of CLCuMuV-Raj and its spread toward the Sindh province where previously Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Shahdadpur virus (CLCuShV) have been reported. The findings of the current study demand future research on CLCuD complex to explore the possible reasons for prevalence in the field and how the virus-host-vector compatible interaction can be broken to develop resistant cultivars.

Microbial influencers and cotton leaf curl disease (CLCuD) susceptibility: a network perspective.

Biotic stresses, such as plant viruses, e.g., cotton leaf curl virus (CLCuV), can alter root-associated and leaf-associated microbial diversities in plants. There are complex ecological dynamics at play, with each microbe contributing to a multitude of biotic and abiotic interactions, thus deciding the stability of the plant's ecosystem in response to the disease. Deciphering these networks of interactions is a challenging task. The inferential research in microbiome is also at a nascent stage, often constrained by the underlying analytical assumptions and the limitations with respect to the depth of sequencing. There is also no real consensus on network-wide statistics to identify the influential microbial players in a network. Guided by the latest developments in network science, including recently published metrics such as Integrated View of Influence (IVI) and some other centrality measures, this study provides an exposé of the most influential nodes in the rhizospheric and phyllospheric microbial networks of the cotton leaf curl disease (CLCuD) susceptible, partially tolerant, and resistant cotton varieties. It is evident from our results that the CLCuD-resistant Gossypium arboreum possesses an equal share of keystone species, which helps it to withstand ecological pressures. In the resistant variety, the phyllosphere harbors the most influential nodes, whereas in the susceptible variety, they are present in the rhizosphere. Based on hubness score, spreading score, and IVI, the top 10 occurring keystone species in the FDH-228 (resistant) variety include Actinokineospora, Cohnella, Thermobacillus, Clostridium, Desulfofarcimen, and MDD-D21. Elusimicrobia, Clostridium-sensu-stricto_12, Candidatus woesebacteria, and Dyella were identified as the most influential nodes in the PFV-1 (partially tolerant) variety. In the PFV-2 (susceptible) variety, the keystone species were identified as Georginia, Nesterenkonia, Elusimicrobia MVP-88, Acetivibrio, Tepedisphaerales, Chelatococcus, Nitrosospira, and RCP2-54. This concept deciphers the diseased and healthy plant's response to viral disease, which may be microbially mediated.

Utilization of primary and secondary biochemical compounds in cotton as diagnostic markers for measuring resistance to cotton leaf curl virus.

Introduction: Cotton (Gossypium hirsutum L.) is one of the most important staple fibrous crops cultivated in India and globally. However, its production and quality are greatly hampered by cotton leaf curl disease (CLCuD) caused by cotton leaf curl virus (CLCuV). Therefore, the aim of the present study was to investigate the biochemical mechanisms associated with CLCuD resistance in contrasting cotton genotypes. Methods: Four commercial cotton varieties with susceptible (HS 6 and RCH-134 BG-II) and resistant (HS 1236 and Bunty) responses were used to analyze the role of primary (sugar, protein, and chlorophyll) and secondary (gossypol, phenol, and tannin) biochemical compounds produced by the plants against infection by CLCuV. The resistant cultivars with increased activity of protein, phenol, and tannin exhibited biochemical barriers against CLCuV infection, imparting resistance in cotton cultivars. Results: Reducing sugar in the healthy plants of the susceptible Bt cultivar RCH 134 BG-II exhibited the highest value of 1.67 mg/g at 90 days. In contrast, the lowest value of 0.07 mg g-1 was observed at 60 DAS in the highly diseased plants of the susceptible hybrid HS 6. Higher phenol content (0.70 mg g-1) was observed at 90 DAS in resistant cultivars, whereas highly susceptible plants exhibited the least phenol (0.25 mg g-1) at 90 DAS. The lowest protein activity was observed at 120 DAS in susceptible cultivars HS 6 (9.4 mg g-1) followed by RCH 134 BG-II (10.5 mg g-1). However, other biochemical compounds, including chlorophyll, sugar, and gossypol, did not show a significant role in resistance against CLCuV. The disease progression analysis in susceptible cultivars revealed non-significant differences between the two susceptible varieties. Discussion: Nevertheless, these compounds are virtually associated with the basic physiological and metabolic mechanisms of cotton plants. Among the primary biochemical compounds, only protein activity was proposed as the first line of defense in cotton against CLCuV. The secondary level of defense line in resistance showed the activity of secondary biochemical compounds phenol and tannins, which displayed a significant increase in their levels while imparting resistance against CLCuV in cotton.

Modeling and analysis for the transmission dynamics of cotton leaf curl virus using fractional order derivatives.

In this study, we examine the Atangana Baleanu Caputo fractional order for the transmission dynamics of the Cotton Leaf Curl Virus disease. The model took into account both cotton plants and vector populations. The existence and uniqueness, positivity and boundedness of the solution to the model, as well as other fundamental concepts, were examined. Additionally, the Ulam-Hyres condition stability of the suggested model was demonstrated using functional techniques. Using the Adams-Bashforth method, the numerical solution for our suggested model was computed. The numerical result shows that the disease spreads more slowly as the fractional order decreases from 1.00 to 0.72.

Cotton leaf segmentation with composite backbone architecture combining convolution and attention.

Plant leaf segmentation, especially leaf edge accurate recognition, is the data support for automatically measuring plant phenotypic parameters. However, adjusting the backbone in the current cutting-edge segmentation model for cotton leaf segmentation applications requires various trial and error costs (e.g., expert experience and computing costs). Thus, a simple and effective semantic segmentation architecture (our model) based on the composite backbone was proposed, considering the computational requirements of the mainstream Transformer backbone integrating attention mechanism. The composite backbone was composed of CoAtNet and Xception. CoAtNet integrated the attention mechanism of the Transformers into the convolution operation. The experimental results showed that our model outperformed the benchmark segmentation models PSPNet, DANet, CPNet, and DeepLab v3+ on the cotton leaf dataset, especially on the leaf edge segmentation (MIoU: 0.940, BIoU: 0.608). The composite backbone of our model integrated the convolution of the convolutional neural networks and the attention of the Transformers, which alleviated the computing power requirements of the Transformers under excellent performance. Our model reduces the trial and error cost of adjusting the segmentation model architecture for specific agricultural applications and provides a potential scheme for high-throughput phenotypic feature detection of plants.

Sentinel plot surveillance of cotton leaf curl disease in Pakistan- a case study at the cultivated cotton-wild host plant interface.

A sentinel plot case study was carried out to identify and map the distribution of begomovirus-betasatellite complexes in sentinel plots and commercial cotton fields over a four-year period using molecular and high-throughput DNA 'discovery' sequencing approaches. Samples were collected from 15 study sites in the two major cotton-producing areas of Pakistan. Whitefly- and leafhopper-transmitted geminiviruses were detected in previously unreported host plant species and locations. The most prevalent begomovirus was cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu). Unexpectedly, a recently recognized recombinant, cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Ra) was prevalent in five of 15 sites. cotton leaf curl Alabad virus (CLCuAlV) and cotton leaf curl Kokhran virus-Kokhran, 'core' members of CLCuD-begomoviruses that co-occurred with CLCuMuV in the 'Multan' epidemic were detected in one of 15 sentinel plots. Also identified were chickpea chlorotic dwarf virus and 'non-core' CLCuD-begomoviruses, okra enation leaf curl virus, squash leaf curl virus, and tomato leaf curl New Delhi virus. Cotton leaf curl Multan betasatellite (CLCuMuB) was the most prevalent CLCuD-betasatellite, and less commonly, two 'non-core' betasatellites. Recombination analysis revealed previously uncharacterized recombinants among helper virus-betasatellite complexes consisting of CLCuKoV, CLCuMuV, CLCuAlV and CLCuMuB. Population analyses provided early evidence for CLCuMuV-Ra expansion and displacement of CLCuKoV-Bu in India and Pakistan from 2012-2017. Identification of 'core' and non-core CLCuD-species/strains in cotton and other potential reservoirs, and presence of the now predominant CLCuMuV-Ra strain are indicative of ongoing diversification. Investigating the phylodynamics of geminivirus emergence in cotton-vegetable cropping systems offers an opportunity to understand the driving forces underlying disease outbreaks and reconcile viral evolution with epidemiological relationships that also capture pathogen population shifts.

qPCR Assay as a Tool for Examining Cotton Resistance to the Virus Complex Causing CLCuD: Yield Loss Inversely Correlates with Betasatellite, Not Virus, DNA Titer.

Cotton leaf curl disease (CLCuD) is a significant constraint to the economies of Pakistan and India. The disease is caused by different begomoviruses (genus Begomovirus, family Geminiviridae) in association with a disease-specific betasatellite. However, another satellite-like molecule, alphasatellite, is occasionally found associated with this disease complex. A quantitative real-time PCR assay for the virus/satellite components causing CLCuD was used to investigate the performance of selected cotton varieties in the 2014-2015 National Coordinated Varietal Trials (NCVT) in Pakistan. The DNA levels of virus and satellites in cotton plants were determined for five cotton varieties across three geographic locations and compared with seed cotton yield (SCY) as a measure of the plant performance. The highest virus titer was detected in B-10 (0.972 ng·µg-1) from Vehari and the lowest in B-3 (0.006 ng·µg-1) from Faisalabad. Likewise, the highest alphasatellite titer was found in B-1 (0.055 ng·µg-1) from Vehari and the lowest in B-1 and B-2 (0.001 ng·µg-1) from Faisalabad. The highest betasatellite titer was found in B-23 (1.156 ng·µg-1) from Faisalabad and the lowest in B-12 (0.072 ng·µg-1) from Multan. Virus/satellite DNA levels, symptoms, and SCY were found to be highly variable between the varieties and between the locations. Nevertheless, statistical analysis of the results suggested that betasatellite DNA levels, rather than virus or alphasatellite DNA levels, were the important variable in plant performance, having an inverse relationship with SCY (-0.447). This quantitative assay will be useful in breeding programs for development of virus resistant plants and varietal trials, such as the NCVT, to select suitable varieties of cotton with mild (preferably no) symptoms and low (preferably no) virus/satellite. At present, no such molecular techniques are used in resistance breeding programs or varietal trials in Pakistan.