Controlling agricultural pests using suitable biocontrol agents has been considered the best strategy for sustainable agriculture. Charcoal rot caused by a necrotrophic fungus Macrophomina phaseolina is responsible for a 30–50% annual reduction in soybean yield worldwide. Little is known about the role of Bacillus clausii in reducing charcoal rot disease severity in the soybean crop. In this study, we investigated plant growth promoting and antagonistic potential of Pseudomonas putida (MT604992) and Bacillus clausii (MT604989) against charcoal rot disease incidence in soybean. Among twenty bacteria isolated from soil and water samples of two different hot springs of Gilgit-Baltistan, Pakistan, 80% were siderophore positive; 65% were hydrogen cyanide (HCN) positive; 55%, 30%, and 75% were phosphate, potassium, and zinc solubilizers, respectively. Based on higher antagonistic activities and plant growth promoting traits five strains were selected for in vitro screening. Out of all tested strains, Pseudomonas putida and Bacillus clausii showed a significant increase in germination, growth, and disease suppression in soybean. These strains produced a pronounced increase in relative water content, photosynthetic pigments, membrane stability, proline, antioxidant enzymes status, phytohormones content (Salicylic acid, and Jasmonic acid), and disease suppression in comparison to control plants. Bacillus clausii mitigated the disease by 97% with a marked increase in the proline content (73% and 89%), superoxide dismutase (356% and 208%), peroxidase (439% and 138.6%), catalase (255.8% and 80.8%), and ascorbate peroxidase (228% and 90%) activities in shoots and roots, respectively. Infected plants showed an increase in salicylic acid and jasmonic acid content which was further increased with the application of the selected strains to increase resistance against pathogens. To our knowledge, this is the first study showing a rise in salicylic acid and jasmonic acid in Macrophomina phaseolina infected plants. These two strains are suggested as a cost-effective, eco-friendly, and sustainable alternative to chemical fungicides. However, there is a need to explore the field testing and molecular mechanisms leading to disease suppression by these strains.

Vascular plant one-zinc-finger (VOZ) transcription factors regulate plant growth and development under drought conditions. Six VOZ transcription factors encoding genes exist in soybean genome (both in Glycine max and Glycine soja). Herein, GmVOZs and GsVOZs were identified through in silico analysis and characterized with different bioinformatics tools and expression analysis. Phylogenetic analysis classified VOZ genes in four groups. Sequence logos analysis among G. max and G. soja amino acid residues revealed higher conservation. Presence of stress related cis-elements in the upstream regions of GmVOZs and GsVOZs highlights their role in tolerance against abiotic stresses. The collinearity analysis identified 14 paralogous/orthologous gene pairs within and between G. max and G. soja. The Ka/Ks values showed that soybean VOZ genes underwent selection pressure with limited functional deviation arising from whole genome and segmental duplication. The GmVOZs and GsVOZs were found to express in roots and leaves at seedling stage. The qRT-PCR revealed that GmVOZs and GsVOZs transcripts can be regulated by abiotic stresses such as polyethylene glycol (PEG). The findings of this study will provide a reference to decipher physiological and molecular functions of VOZ genes in soybean.

The fibrillin (FBN) gene family is widely distributed in all photosynthetic organisms. Members of this gene family are involved in plant growth and development and their response to various biotic and abiotic stress factors. In this study, 16 members of FBN were identified in Glycine max and characterized by using different bioinformatics tools. Phylogenetic analysis classified FBN genes into seven groups. The presence of stress-related cis-elements in the upstream region of GmFBN highlighted their role in tolerance against abiotic stresses. To further decipher the function, physiochemical properties, conserved motifs, chromosomal localization, subcellular localization, and cis-acting regulatory elements were also analyzed. Gene expression analysis based on FPKM values revealed that GmFBNs greatly enhanced soybean drought tolerance and controlled the expression of several genes involved in drought response, except for GmFBN-4, GmFBN-5, GmFBN-6, GmFBN-7 and GmFBN-9. For high throughput genotyping, an SNP-based CAPS marker was also developed for the GmFBN-15 gene. The CAPS marker differentiated soybean genotypes based on the presence of either the GmFBN-15-G or GmFBN-15-A alleles in the CDS region. Association analysis showed that G. max accessions containing the GmFBN-15-A allele at the respective locus showed higher thousand seed weight compared to accessions containing the GmFBN-15-G allele. This research has provided the basic information to further decipher the function of FBN in soybean.

Cysteine-rich poly comb-like protein (CPP) is a member of cysteine-rich transcription factors that regulates plant growth and development. In the present work, we characterized twelve CPP transcription factors encoding genes in soybean (Glycine max). Phylogenetic analyses classified CPP genes into six clades. Sequence logos analyses between G. max and G. soja amino acid residues exhibited high conservation. The presence of growth and stress-related cis-acting elements in the upstream regions of GmCPPs highlight their role in plant development and tolerance against abiotic stress. Ka/Ks levels showed that GmCPPs experienced limited selection pressure with limited functional divergence arising from segmental or whole genome duplication events. By using the PAN-genome of soybean, a single nucleotide polymorphism was identified in GmCPP-6. To perform high throughput genotyping, a kompetitive allele-specific PCR (KASP) marker was developed. Association analyses indicated that GmCPP-6-T allele of GmCPP-6 (in exon region) was associated with higher thousand seed weight under both water regimes (well-water and water-limited). Taken together, these results provide vital information to further decipher the biological functions of CPP genes in soybean molecular breeding.

Determination of Effective Method of NPK Fertilization in Pea (Pisum sativum L.) Cultivars Grown in Pakistan