Tissue specific expression of bacterial cellulose synthase (Bcs) genes improves cotton fiber length and strength.

Fiber growing inside the cotton bolls is a highly demandable product and its quality is key to the success of the textile industry. Despite the various efforts to improve cotton fiber staple length Pakistan has to import millions of bales to sustain its industrial needs. To improve cotton fiber quality Bacterial cellulose synthase (Bcs) genes (acsA, acsB) were expressed in a local cotton variety CEMB-00. In silico studies revealed a number of conserved domains both in the cotton-derived and bacterial cellulose synthases which are essential for the cellulose synthesis. Transformation efficiency of 1.27% was achieved by using Agrobacterium shoot apex cut method of transformation. The quantitative mRNA expression analysis of the Bcs genes in transgenic cotton fiber was found to be many folds higher during secondary cell wall synthesis stage (35 DPA) than the expression during elongation phase (10 DPA). Average fiber length of the transgenic cotton plant lines S-00-07, S-00-11, S-00-16 and S-00-23 was calculated to be 13.02% higher than that of the non-transgenic control plants. Likewise, the average fiber strength was found to be 20.92% higher with an enhanced cellulose content of 22.45%. The mutated indigenous cellulose synthase genes of cotton generated through application of CRISPR/Cas9 resulted in 6.03% and 12.10% decrease in fiber length and strength respectively. Furthermore, mature cotton fibers of transgenic cotton plants were found to have increased number of twists with smooth surface as compared to non-transgenic control when analyzed under scanning electron microscope. XRD analysis of cotton fibers revealed less cellulose crystallinity index in transgenic cotton fibers as compared to control fibers due to deposition of more amorphous cellulose in transgenic fibers as a result of Bcs gene expression. This study paved the way towards unraveling the fact that Bcs genes influence cellulose synthase activity and this enzyme helps in determining the fate of cotton fiber length and strength.

Discovery of succinate dehydrogenase candidate fungicides via lead optimization for effective resistance management of Fusarium oxysporum f. sp. capsici.

Fusarium wilt of chili caused by the fungus Fusarium oxysporum f. sp. capsici (FCO) severely reduces the production of chili worldwide. There is growing evidence of resistance to commercial fungicides targeting succinate dehydrogenase (Sdh) of FCO soliciting the development of new Sdh inhibitors (SdhIs). In the current work, optimized docking and virtual screening were used to mine twelve SdhIs from the ZINC database, followed by in vitro antifungal evaluation on spore and radial mycelium development. Four new promising SdhIs exhibiting a mean mycelium inhibition rate greater than 85.6% (F = 155.8, P = 0.001, P < 0.05) were observed on ten strains of virulent and resistant FCO. Importantly, three of the discovered molecules exhibited potent spore germination inhibition (≥ 80%, P = 0.01, P < 0.05) compared to the commonly used fungicide penthiopyrad. A significant positive correlation (r* ≥ 0.67, P < 0.05) between the activities of the newly discovered SdhIs compared to penthiopyrad against all tested FCO strains indicated a broad-spectrum fungicidal activity. The current findings indicate that the four SdhI's discovered could judiciously replace certain commercial SdhIs that some FCO displays resistance to. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03157-8.

Droplet-based microfluidics platform for antifungal analysis against filamentous fungi.

Fungicides are extensively used in agriculture to control fungal pathogens which are responsible for significant economic impact on plant yield and quality. The conventional antifungal screening techniques, such as water agar and 96-well plates, are based on laborious protocols and bulk analysis, restricting the analysis at the single spore level and are time consuming. In this study, we present a droplet-based microfluidic platform that enables antifungal analysis of single spores of filamentous fungus Alternaria alternata. A droplet-based viability assay was developed, allowing the germination and hyphal growth of single A. alternata spores within droplets. The viability was demonstrated over a period of 24 h and the antifungal screening was achieved using Kunshi/Tezuma as antifungal agent. The efficacy results of the droplet-based antifungal analysis were compared and validated with the results obtained from conventional protocols. The percentage inhibitions assessed by the droplet-based platform were equivalent with those obtained by the other two methods, and the Pearson correlation analysis showed high correlation between the three assays. Taken together, this droplet-based microfluidic platform provides a wide range of potential applications for the analysis of fungicide resistance development as well as combinatorial screening of other antimicrobial agents and even antagonistic fungi.

Expression of ice recrystallization inhibition protein in transgenic potato lines associated with reduced electrolyte leakage and efficient recovery post freezing injury.

Potato (Solanum tuberosum L.) is considered to be frost-susceptible as short spells of frost can reduce the tuber yield and quality. Ice recrystallization inhibition (IRI) protein helps prevent growth of ice crystals in the cell apoplast during frost and help prevent damage associated with freezing stress. In this study, we investigated the in planta potential of Lolium perenne derived IRI3 transgene in improving the tolerance of transgenic potato lines for freezing stress. The codon optimized IRI3 transgene was introduced into potato cultivar Diamant through Agrobacterium mediated transformation. Three transgenic potato lines were successfully generated which were confirmed for transgene insertion and genomic integration by polymerase chain reaction and Southern blot. It was evident that the IRI3 transcript decreased in initial 24 h of cold stress treatment while the IRI3 mRNA expression up regulated in subsequent hours of cold treatment with maximum increase to 20 folds at 96 h post stress. A similar trend was also revealed in ion-leakage assay which showed that during cold stress, the transgenic potato lines depicted reduced ion leakage of 14-22% as compared to non-transgenic control plants. Further, the generated transgenic potato lines were tolerant to the frost spell in quarantine field conditions as compared to the non-transgenic potato lines. Additionally, the transgenic lines exhibited efficient recovery post frost injury in field conditions. The biochemical profiles of chlorophyll, proline and higher levels of antioxidant enzyme (superoxide dismutase, Catalase) activity and malondialdehyde levels showed that despite the phenotypic impact of low temperature, the transgenic potato lines quickly adjusted to maintain their cellular homeostasis post freezing stress by increasing the antioxidant defenses. This study suggests that up regulation of IRI3 transcript and regulatory network of cold stress response in transgenic potato lines improve frost tolerance and help stabilize yield in cultivated potato.

Chitinase genes from Metarhizium anisopliae for the control of whitefly in cotton.

Entomopathogenic fungi produces endochitianses, involved in the degradation of insect chitin to facilitate the infection process. Endochitinases (Chit1) gene of family 18 glycosyl hydrolyses were amplified, cloned and characterized from genomic DNA of two isolates of Metarhizium anisopliae. Catalytic motif of family 18 glycosyl hydrolyses was found in Chit1 of M. anisopliae, while no signal peptide was found in any isolate, whereas substrate-binding motif was found in Chit1 of both isolates. Phylogenetic analysis revealed the evolutionary relationship among the fungal chitinases of Metarhizium. The Chit1 amplified were closely related to the family 18 glycosyl hydrolyses. Transient expressions of Chit1 in cotton plants using Geminivirus-mediated gene silencing vector of Cotton Leaf Crumple Virus (CLCrV) revealed the chitinase activity of Chit1 genes amplified from both of the isolates of M. anisopliae when compared with the control. Transformed cotton plants were virulent against fourth instar nymphal and adult stages of Bemisia tabaci which resulted in the mortality of both fourth instar nymphal and adult B. tabaci. Thus, the fungal chitinases expressed in cotton plants played a vital role in plant defence against B. tabaci. However, further studies are required to explore the comparative effectiveness of chitinases from different fungal strains against economically important insect pests.

Biotechnological application of endophytic filamentous bipolaris and curvularia: a review on bioeconomy impact.

The filamentous Bipolaris and Curvularia genera consist of species known to cause severe diseases in plants and animals amounting to an estimated annual loss of USD $10 billion worldwide. Despite the harmful effect of Bipolaris and Curvularia species, scarce attention is paid on beneficial areas where the fungi are used in industrial processes to generate biotechnological products. Catalytic potential of Bipolaris and Curvularia species in the production of biodiesel, bioflucculant, biosorbent, and mycoherbicide are promising for the bioeconomy. It is herein demonstrated that knowledge-based application of some endophytic Bipolaris and Curvularia species are indispensable vectors of sustainable economic development. In the twenty-first century, India, China, and the USA have taken progress in the biotechnological application of these fungi to generate wealth. As such, some Bipolaris and Curvularia species significantly impact on global crop improvement, act as catalyst in batch-reactors for biosynthesis of industrial enzymes and medicines, bioengineer of green-nanoparticle, agent of biofertilizer, bioremediation and bio-hydrometallurgy. For the first time, this study discusses the current advances in biotechnological application of Bipolaris and Curvularia species and provide new insights into the prospects of optimizing their bioengineering potential for developing bioeconomy.

Global challenges faced by engineered Bacillus thuringiensis Cry genes in soybean (Glycine max L.) in the twenty-first century.

The most important insect pests causing severe economic damages to soybean (Glycine max L.) production worldwide are Chrysodeixis includens (Walker, Noctuidae), Anticarsia gemmatalis (Hübner, Erebidae), Helicoverpa gelotopoeon (Dyar, Noctuidae), Crocidosema aporema (Walsingham; Tortricidae), Spodoptera albula (Walker, Noctuidae), S. cosmiodes (Walker, Noctuidae), S. eridania (Stoll, Noctuidae), S. frugiperda (Smith; Noctuidae), Helicoverpa armigera (Hübner, Noctuidae), H. zea (Boddie; Noctuidae) and Telenomus podisi (Hymenoptera,Platygastidae). Despite the success of biotech Bacillus thuringiensis (Bt)/herbicide tolerance (HT)-soybean in the past decade in terms of output, unforeseen mitigated performances have been observed due to changes in climatic events that favors the emergence of insect resistance. Thus, there is a need to develop hybrids with elaborated gene stacking to avert the upsurge in insect field tolerance to crystal (Cry) toxins in Bt-soybean. This study covers the performance of important commercial transgenic soybean developed to outwit destructive insects. New gene stacking soybean events such as Cry1Ac-, Cry1AF- and PAT-soybean (DAS-81419-2®, Conkesta™ technology), and MON-87751-7 × MON-87701-2 × MON 87708 × MON 89788 (bearing Cry1A.105 [Cry1Ab, Cry1F, Cry1Ac], Cry2Ab, Cry1Ac) are being approved and deployed in fields. Following this deployment trend, we recommend herein that plant-mediated RNA interference into Bt-soybean, and the application of RNA-based pesticides that is complemented by other best agricultural practices such as refuge compliance, and periodic application of low-level insecticides could maximize trait durability in Bt-soybean production in the twenty-first century.

Evidence of genetically diverse virulent mating types of Phytophthora capsici from Capsicum annum L.

Chili pepper (Capsicum annum L.) is an important economic crop that is severely destroyed by the filamentous oomycete Phytophthora capsici. Little is known about this pathogen in key chili pepper farms in Punjab province, Pakistan. We investigated the genetic diversity of P. capsici strains using standard taxonomic and molecular tools, and characterized their colony growth patterns as well as their disease severity on chili pepper plants under the greenhouse conditions. Phylogenetic analysis based on ribosomal DNA (rDNA), ß-tubulin and translation elongation factor 1α loci revealed divergent evolution in the population structure of P. capsici isolates. The mean oospore diameter of mating type A1 isolates was greater than that of mating type A2 isolates. We provide first evidence of an uneven distribution of highly virulent mating type A1 and A2 of P. capsici that are insensitive to mefenoxam, pyrimorph, dimethomorph, and azoxystrobin fungicides, and represent a risk factor that could ease outpacing the current P. capsici management strategies.

Global invasive Cochliobolus species: cohort of destroyers with implications in food losses and insecurity in the twenty-first century.

Matching the global food demand by 2050 and to ensure the stability of food security in over than 99 countries, it is necessary to scale up the production of food such as sorghum, wheat, rice, maize and sugarcane which are however natural hosts of Cochliobolus species. Cochliobolus species major epidemics such as the Great Bengal famine, Southern corn leaf blight, and Northern leaf spot blight were associated with substantial economic losses in the past decades. Thus, there is an urgent need to establish a specific coordinated global surveillance program for the migration of invasive Cochliobolus species, planning contextual control programs engaging all agricultural stakeholders and information sharing in real time for prevention of disastrous Cochliobolus disease outbreak effects. We discuss pertinent outcome of interactions of cash crops with Cochliobolus species having devastating impact on the livelihood of farmers and food security. While post-genomic era elucidated prominent differences among Cochliobolus heterostrophus, C. carbonum, C. victoriae, C. lunatus and C. miyabeanus, their destructive potentials and implications in food losses remained unearthed. Intriguingly, the annual colossal losses caused by Cochliobolus species in the production perspective of sorghum, wheat, rice, maize, cassava and soybean is estimated over 10 billion USD worldwide. This paper provides a comprehensive analysis of the invasive Cochliobolus species distribution and diversity, evolving pathogenicity, persistent diseases, threats and epidemics, consequences on food crops production and increasing global food insecurity issues.

Discovering Novel Alternaria solani Succinate Dehydrogenase Inhibitors by in Silico Modeling and Virtual Screening Strategies to Combat Early Blight.

Alternaria blight is an important foliage disease caused by Alternaria solani. The enzyme Succinate dehydrogenase (SDH) is a potential drug target because of its role in tricarboxylic acid cycle. Hence targeting Alternaria solani SDH enzyme could be efficient tool to design novel fungicides against A. solani. We employed computational methodologies to design new SDH inhibitors using homology modeling; pharmacophore modeling and structure based virtual screening. The three dimensional SDH model showed good stereo-chemical and structural properties. Based on virtual screening results twelve commercially available compounds were purchased and tested in vitro and in vivo. The compounds were found to inhibit mycelial growth of A. solani. Moreover in vitro trials showed that inhibitory effects were enhanced with increase in concentrations. Similarly increased disease control was observed in pre-treated potato tubers. Hence the applied in silico strategy led us to identify novel fungicides.

Invasive Aspergillus terreus morphological transitions and immunoadaptations mediating antifungal resistance.

BACKGROUND AND AIMS: Aspergillus terreus Thom is a pathogen of public health and agricultural importance for its seamless abilities to expand its ecological niche. The aim of this study was holistically to investigate A. terreus morphological and immunoadaptations and their implication in antifungal resistance and proliferation during infection. MATERIALS AND METHODS: In-depth unstructured mining of relevant peer-reviewed literature was performed for A. terreus morphological, immune, resistance, and genetic diversity based on the sequenced calmodulin-like gene. RESULTS: Accessory conidia and phialidic conidia produced by A. terreus confer discrete anti-fungal resistance that ensures survivability during therapies. Interestingly, by producing unique metabolites such as Asp-melanin and terretonin, A. terreus is capable of hijacking macrophages and scavenging iron, respectively. As such, A. terreus has established a rare mechanism to mitigate phagocytosis and swing the interaction dynamics in favor of its proliferation and survival in hosts. CONCLUSION: It is further unraveled that besides A. terreus genetic diversity, morphological, biochemical, and immunologic adaptations associated with conidia germination and discharge of chemical signals during infection enable masking of the host defense as an integral part of its strategy to survive and rapidly colonize hosts.