Bacillus altitudinis HNH7 and Bacillus velezensis HNH9 promote plant growth through upregulation of growth-promoting genes in upland cotton.

AIMS: The potential of endophytic Bacillus strains to improve plant growth and yield was evaluated. METHODS AND RESULTS: Endophytic Bacillus altitudinis HNH7 and Bacillus velezensis HNH9 were evaluated for their growth-promoting traits. In an in vitro plate assay, HNH7 and HNH9 exhibited proteolytic, amylolytic, lipolytic and cellulolytic activity. HNH7 and HNH9 were able to solubilize iron by producing siderophores but were unable to solubilize insoluble phosphate. PCR confirmed the presence of four growth-promoting genes viz. pvd, budA, asbA and satA in the genome of HNH7, while HNH9 also possessed the same genes except for budA. In a greenhouse experiment, HNH7 and HNH9 promoted the growth of upland cotton plants by upregulating the expression of growth-linked genes, EXP6, ARF1, ARF18, IAA9, CKX6 and GID1b. However, the expression of genes involved in ethylene biosynthesis, that is ERF and ERF17 was downregulated after treating the plants with HNH7 and HNH9 compared to the control. Furthermore, cotton plants treated with HNH7 and HNH9 exhibited a significantly higher rate of photosynthesis and stomatal conductance. CONCLUSION: HNH7 and HNH9 showed a promising potential to promote the growth of cotton plants. SIGNIFICANCE AND IMPACT OF STUDY: Research on plant growth-promoting Bacillus strains can lead to the formation of biofertilizers.

Novel Genetic Dysregulations and Oxidative Damage in Fusarium graminearum Induced by Plant Defense Eliciting Psychrophilic Bacillus atrophaeus TS1.

This study elaborates inter-kingdom signaling mechanisms, presenting a sustainable and eco-friendly approach to combat biotic as well as abiotic stress in wheat. Fusarium graminearum is a devastating pathogen causing head and seedling blight in wheat, leading to huge yield and economic losses. Psychrophilic Bacillus atrophaeus strain TS1 was found as a potential biocontrol agent for suppression of F. graminearum under low temperature by carrying out extensive biochemical and molecular studies in comparison with a temperate biocontrol model strain Bacillus amyloliquefaciens FZB42 at 15 and 25 °C. TS1 was able to produce hydrolytic extracellular enzymes as well as antimicrobial lipopeptides, i.e., surfactin, bacillomycin, and fengycin, efficiently at low temperatures. The Bacillus strain-induced oxidative cellular damage, ultrastructural deformities, and novel genetic dysregulations in the fungal pathogen as the bacterial treatment at low temperature were able to downregulate the expression of newly predicted novel fungal genes potentially belonging to necrosis inducing protein families (fgHCE and fgNPP1). The wheat pot experiments conducted at 15 and 25 °C revealed the potential of TS1 to elicit sudden induction of plant defense, namely, H2O2 and callose enhanced activity of plant defense-related enzymes and induced over-expression of defense-related genes which accumulatively lead to the suppression of F. graminearum and decreased diseased leaf area.

Genomic Features and Molecular Function of a Novel Stress-Tolerant Bacillus halotolerans Strain Isolated from an Extreme Environment.

Due to its topographical position and climatic conditions, the Qinghai-Tibet Plateau possesses abundant microorganism resources. The extremophilic strain KKD1 isolated from Hoh Xil possesses strong stress tolerance, enabling it to propagate under high salinity (13%) and alkalinity (pH 10.0) conditions. In addition, KKD1 exhibits promising biocontrol activity against plant pathogens. To further explore these traits at the genomic level, we performed whole-genome sequencing and analysis. The taxonomic identification according to the average nucleotide identity based on BLAST revealed that KKD1 belongs to Bacillus halotolerans. Genetic screening of KKD1 revealed that its stress resistance mechanism depends on osmotic equilibrium, membrane transportation, and the regulation of ion balance under salt and alkaline stress. The expression of genes involved in these pathways was analyzed using real-time quantitative PCR. The presence of different gene clusters encoding antimicrobial secondary metabolites indicated the various pathways by which KKD1 suppresses phytopathogenic growth. Moreover, the lipopeptides surfactin and fengycin were identified as being significant antifungal components of KKD1. Through comparative genomics analysis, we noticed that KKD1 harbored specific genes involved in stress resistance and biocontrol, thus providing a new perspective on the genomic features of the extremophilic Bacillus species.

Nematicidal Volatiles from Bacillus atrophaeus GBSC56 Promote Growth and Stimulate Induced Systemic Resistance in Tomato against Meloidogyne incognita.

Bacillus volatiles to control plant nematodes is a topic of great interest among researchers due to its safe and environmentally friendly nature. Bacillus strain GBSC56 isolated from the Tibet region of China showed high nematicidal activity against M. incognita, with 90% mortality as compared with control in a partition plate experiment. Pure volatiles produced by GBSC56 were identified through gas chromatography and mass spectrometry (GC-MS). Among 10 volatile organic compounds (VOCs), 3 volatiles, i.e., dimethyl disulfide (DMDS), methyl isovalerate (MIV), and 2-undecanone (2-UD) showed strong nematicidal activity with a mortality rate of 87%, 83%, and 80%, respectively, against M. incognita. The VOCs induced severe oxidative stress in nematodes, which caused rapid death. Moreover, in the presence of volatiles, the activity of antioxidant enzymes, i.e., SOD, CAT, POD, and APX, was observed to be enhanced in M. incognita-infested roots, which might reduce the adverse effect of oxidative stress-induced after infection. Moreover, genes responsible for plant growth promotion SlCKX1, SlIAA1, and Exp18 showed an upsurge in expression, while AC01 was downregulated in infested plants. Furthermore, the defense-related genes (PR1, PR5, and SlLOX1) in infested tomato plants were upregulated after treatment with MIV and 2-UD. These findings suggest that GBSC56 possesses excellent biocontrol potential against M. incognita. Furthermore, the study provides new insight into the mechanism by which GBSC56 nematicidal volatiles regulate antioxidant enzymes, the key genes involved in plant growth promotion, and the defense mechanism M. incognita-infested tomato plants use to efficiently manage root-knot disease.

First report of post-harvest Fusarium rot of mandarin Citrus reticulata cv. 'Kinnow' caused by Fusarium equiseti in Pakistan.

Citrus reticulata cv. 'Kinnow' mandarin is the most popular and widely grown fruit crop in Pakistan. During 2017, a survey was conducted to the local citrus fruit markets of Faisalabad, Pakistan. Citrus fruits (n=50) exhibiting stem end rot and fruit rot were collected with 15% disease incidence. The stem end region showed light to dark brown lesions and white fungal growth was also observed in the severely infected fruit. Infected fruit were excised into 2mm2 segments, surface disinfected with 1% NaClO, rinsed with sterilized water and dried. Later, these tissues were placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Purified isolates produced white colonies with beige pigmentation. The frequency of fungal isolation was 47%. Microscopic observations revealed that macroconidia (n=50) had 5 to 6 septations, with a prominent dorsiventral curvature, tapered and elongated apical cell, and a foot shape basal cell. The macroconidia were measuring 22 to 45 × 2.9 to 4.3 µm with an average of 31 × 3.6 µm. However, microconidia were not observed. Chlamydospores were globose, intercalary, solitary, or in pairs, appearing in chains (Leslie and Summerell 2006). For molecular identification, DNA was extracted from all isolates. The internal transcribed spacer region (ITS) ITS1/4 (White et al. 1990), translation elongation factor-1 alpha (TEF) EF1/2 (O'Donnell et al. 1998), and RNA polymerase II subunit 1 (RPB1) (O'Donnell et al. 2013) were amplified using PCR and the product was subsequently sequenced. Based on BLAST analysis, the isolate was identified as Fusarium equiseti (FUS-21). The sequences of the representative isolate FUS-21 were deposited in the GenBank with accession numbers (ITS, MH581300), (TEF, MK203749), and (RPB1, MW596599) showing more than 99% similarity with ITS accession GQ505683, TEF accession GQ505594, and 100% to RPB1 accession JX171481. To determine the pathogenicity, 40 healthy surface disinfested citrus fruit were taken. The fruit were inoculated by creating artificial wounds on the surface with a sterilized needle and 10 µL of 105 spores/mL was deposited in the wounds. In case of control fruit were inoculated with 10 µL sterilized distilled water only, and incubated at 25 °C. All fruit inoculated with the putative pathogen, developed symptoms like the original fruit from which they were isolated. The pathogenicity test was repeated twice. Visible white mycelium appeared at the stem end region and the fruits became dried as the infection progressed. However, the control fruit remained asymptomatic. The pathogen was re-isolated from infected fruit and identified based on morphometric and molecular analysis. Previously we have reported F. oxysporum causing citrus fruit rot in Pakistan (Moosa et al. 2020). This is the first report of F. equiseti causing post-harvest rot of citrus fruits in Pakistan. Kinnow is an important fruit crop of Pakistan with huge export value the management of Fusarium rot is quite important to save the loss of fresh produce.

Antagonistic Potential of Novel Endophytic Bacillus Strains and Mediation of Plant Defense against Verticillium Wilt in Upland Cotton.

Verticillium wilt caused by Verticillium dahliae is a threatening disease of cotton, causing economic loss worldwide. In this study, nine endophytic Bacillus strains isolated from cotton roots exhibited inhibitory activity against V. dahliae strain VD-080 in a dual culture assay. B. altitudinis HNH7 and B. velezensis HNH9 were chosen for further experiments based on their high antagonistic activity. The secondary metabolites of HNH7 and HNH9 also inhibited the growth of VD-080. Genetic marker-assisted detection revealed the presence of bacillibactin, surfactin, bacillomycin and fengycin encoding genes in the genome of HNH7 and HNH9 and their corresponding gene products were validated through LC-MS. Scanning electron microscopy revealed mycelial disintegration, curling and shrinkage of VD-080 hyphae after treatment with methanolic extracts of the isolated endophytes. Furthermore, a significant reduction in verticillium wilt severity was noticed in cotton plants treated with HNH7 and HNH9 as compared to control treatments. Moreover, the expression of defense-linked genes, viz., MPK3, GST, SOD, PAL, PPO and HMGR, was considerably higher in plants treated with endophytic Bacillus strains and inoculated with VD-080 as compared to control.

Transcriptional Profiling of Diffusible Lipopeptides and Fungal Virulence Genes During Bacillus amyloliquefaciens EZ1509-Mediated Suppression of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating necrotrophic pathogen that infects multiple crops, and its control is an unremitting challenge. In this work, we attempted to gain insights into the pivotal role of lipopeptides (LPs) in the antifungal activity of Bacillus amyloliquefaciens EZ1509. In a comparative study involving five Bacillus strains, B. amyloliquefaciens EZ1509 harboring four LPs biosynthetic genes (viz. surfactin, iturin, fengycin, and bacilysin) exhibited promising antifungal activity against S. sclerotiorum in a dual-culture assay. Our data demonstrated a remarkable upsurge in LPs biosynthetic gene expression through quantitative reverse transcription PCR during in vitro interaction assay with S. sclerotiorum. Maximum upregulation in LPs biosynthetic genes was observed on the second and third days of in vitro interaction, with iturin and fengycin being the highly expressed genes. Subsequently, Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis confirmed the presence of LPs in the inhibition zone. Scanning electron microscope analysis showed disintegration, shrinkage, plasmolysis, and breakdown of fungal hyphae. During in planta evaluation, S. sclerotiorum previously challenged with EZ1509 showed significant suppression in pathogenicity on detached leaves of tobacco and rapeseed. The oxalic acid synthesis was also significantly reduced in S. sclerotiorum previously confronted with antagonistic bacterium. The expression of major virulence genes of S. sclerotiorum, including endopolygalacturonase-3, oxalic acid hydrolase, and endopolygalacturonase-6, was significantly downregulated during in vitro confrontation with EZ1509.

The Type III Accessory Protein HrpE of Xanthomonas oryzae pv. oryzae Surpasses the Secretion Role, and Enhances Plant Resistance and Photosynthesis.

Many species of plant-pathogenic gram-negative bacteria deploy the type III (T3) secretion system to secrete virulence components, which are mostly characteristic of protein effectors targeting the cytosol of the plant cell following secretion. Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen causing bacterial blight disease, uses the T3 accessory protein HrpE to assemble the pilus pathway, which in turn secretes transcription activator-like (TAL) effectors. The hrpE gene can execute extensive physiological and pathological functions beyond effector secretion. As evidenced in this study, when the hrpE gene was deleted from the Xoo genome, the bacteria incur seriouimpairments in multiplication, motility, and virulence. The virulence nullification is attributed to reduced secretion and translocation of PthXo1, which is a TAL effector that determines the bacterial virulence in the susceptible rice varieties. When the HrpE protein produced by prokaryotic expression is applied to plants, the recombinant protein is highly effective at inducing the defense response. Moreover, leaf photosynthesis efficiency is enhanced in HrpE-treated plants. These results provide experimental avenues to modulate the plant defense and growth tradeoff by manipulating a bacterial T3 accessory protein.

Suppression of Sclerotinia sclerotiorum by the Induction of Systemic Resistance and Regulation of Antioxidant Pathways in Tomato Using Fengycin Produced by Bacillus amyloliquefaciens FZB42.

Lipopeptides from Bacillus species exhibit promising biological control activity against plant pathogens. This study aimed to explore the potential of purified fengycin to induce systemic resistance in tomato against Sclerotinia sclerotiorum. Bacillus amyloliquefaciens FZB42, its mutant AK1S, and their corresponding metabolites showed in vitro inhibition of S. sclerotiorum mycelium. Fengycin derived from an AK1S mutant was purified and identified through HPLC and MALDI-TOF-MS, respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed structural deformities in the fungal mycelium. Moreover, fengycin induced the accumulation of reactive oxygen species (ROS) in S. sclerotiorum mycelium and downregulated the expression of ROS-scavenging genes viz., superoxide dismutase (SsSOD1), peroxidase (SsPO), and catalase (SsCAT1) compared to the untreated control. Furthermore, the lesion size was dramatically reduced in fengycin-treated tomato plants compared to plants infected with S. sclerotiorum only in a greenhouse experiment. Additionally, the transcriptional regulation of defense-related genes GST, SOD, PAL, HMGR, and MPK3 showed the highest upsurge in expression at 48 h post-inoculation (hpi). However, their expression was subsequently decreased at 96 hpi in fengycin + S. sclerotiorum treatment compared to the plants treated with fengycin only. Conversely, the expression of PPO increased in a linear manner up to 96 hpi.

Genetic Screening and Expression Analysis of Psychrophilic Bacillus spp. Reveal Their Potential to Alleviate Cold Stress and Modulate Phytohormones in Wheat.

Abiotic stress in plants pose a major threat to cereal crop production worldwide and cold stress is also notorious for causing a decrease in plant growth and yield in wheat. The present study was designed to alleviate cold stress on plants by inoculating psychrophilic PGPR bacteria belonging to Bacillus genera isolated from extreme rhizospheric environments of Qinghai-Tibetan plateau. The genetic screening of psychrophilic Bacillus spp. CJCL2, RJGP41 and temperate B. velezensis FZB42 revealed presence of genetic features corresponding to cold stress response, membrane transport, signal transduction and osmotic regulation. Subsequently, the time frame study for the expression of genes involved in these pathways was also significantly higher in psychrophilic strains as analyzed through qPCR analysis at 4 ℃. The inoculated cold tolerant Bacillus strains also aided in inducing stress response in wheat by regulating abscisic acid, lipid peroxidation and proline accumulation pathways in a beneficial manner. Moreover, during comparative analysis of growth promotion in wheat all three Bacillus strains showed significant results at 25 ℃. Whereas, psychrophilic Bacillus strains CJCL2 and RJGP41 were able to positively regulate the expression of phytohormones leading to significant improvement in plant growth under cold stress.

Fengycin Produced by Bacillus amyloliquefaciens FZB42 Inhibits Fusarium graminearum Growth and Mycotoxins Biosynthesis.

Fusarium graminearum is a notorious pathogen that causes Fusarium head blight (FHB) in cereal crops. It produces secondary metabolites, such as deoxynivalenol, diminishing grain quality and leading to lesser crop yield. Many strategies have been developed to combat this pathogenic fungus; however, considering the lack of resistant cultivars and likelihood of environmental hazards upon using chemical pesticides, efforts have shifted toward the biocontrol of plant diseases, which is a sustainable and eco-friendly approach. Fengycin, derived from Bacillus amyloliquefaciens FZB42, was purified from the crude extract by HPLC and further analyzed by MALDI-TOF-MS. Its application resulted in structural deformations in fungal hyphae, as observed via scanning electron microscopy. In planta experiment revealed the ability of fengycin to suppress F. graminearum growth and highlighted its capacity to combat disease incidence. Fengycin significantly suppressed F. graminearum, and also reduced the deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and zearalenone (ZEN) production in infected grains. To conclude, we report that fengycin produced by B. amyloliquefaciens FZB42 has potential as a biocontrol agent against F. graminearum and can also inhibit the mycotoxins produced by this fungus.

Volatile Compounds of Endophytic Bacillus spp. have Biocontrol Activity Against Sclerotinia sclerotiorum.

To develop an effective biological agent to control Sclerotinia sclerotiorum, three endophytic Bacillus spp. strains with high antagonistic activity were isolated from maize seed and characterized. In vitro assays revealed that the Bacillus endophytes could produce volatile organic compounds (VOC) that reduced sclerotial production and inhibited mycelial growth of S. sclerotiorum. Gas chromatography-mass spectrometry revealed that the selected strains produced 16 detectable VOC. Eight of the produced VOC exhibited negative effects on S. sclerotiorum, while a further four induced accumulation of reactive oxygen species in mycelial cells. A mixture of VOC produced by Bacillus velezensis VM11 caused morphological changes in the ultrastructure and organelle membranes of S. sclerotiorum mycelial cells. The bromophenol blue assay revealed a yellow color of untreated fungal mycelium, which grew faster and deeper from 24 to 72 h postinoculation, as an indication of reduced pH. The potassium permanganate (KMnO4) titration assay showed that the rate of oxalic acid accumulation was higher in minimal salt liquid medium cultures inoculated with untreated fungal plugs compared with the Bacillus VOC-treated ones. Interestingly, biological control assays using host-plant leaves challenged with treated fungal mycelial plugs produced reduced lesions compared with the control. These findings provide new viable possibilities of controlling diseases caused by S. sclerotiorum using VOC produced by Bacillus endophytes.