Role of endophytic Penicillium species and Pseudomonas monteilii in inducing the systemic resistance in okra against root rotting fungi and their effect on some physiochemical properties of okra fruit.

AIMS: The efficacy of three isolates of endophytic Penicillium species that have shown significant suppressive effect on root rotting fungi in our previous study were further evaluated in pots and field plot experiments for their effect on root diseases of okra, induction of systemic resistance and physiochemical properties of okra fruit. METHODS AND RESULTS: Aqueous suspensions of endophytic Penicillium and Pseudomonas monteilii were applied in pots and field plots using okra as test plant. Data on the fungal infection of roots, plant growth, plant resistance markers like polyphenol, salicylic acid and antioxidant status of plant were determined. These isolates significantly suppressed root diseases and induced systemic resistance via increasing level of resistance markers, polyphenol and salicylic acid besides improving antioxidant activity of Penicillium and P. monteilii-treated plants as compared to control plants. GC-MS analysis of n-hexane extract of mycelium of P. nigricans revealed the presence of 15 different volatile compounds. CONCLUSIONS: Endophytic Penicillium and P. monteilii have potential against root-infecting fungi of okra and can improve plant growth and yield. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic Penicillium species and P. monteilii can suppress root rotting fungi by direct mechanism or induction of systemic resistance in plants.

Seed biopriming with P- and K-solubilizing Enterobacter hormaechei sp. improves the early vegetative growth and the P and K uptake of okra (Abelmoschus esculentus) seedling.

Limited information is available that seed biopriming with plant growth-promoting Enterobacter spp. play a prominent role to enhance vegetative growth of plants. Contrary to Enterobacter cloacae, Enterobacter hormaechei is a less-studied counterpart despite its vast potential in plant growth-promotion mainly through the inorganic phosphorus (P) and potassium (K) solubilization abilities. To this end, 18 locally isolated bacterial pure cultures were screened and three strains showed high P- and K-solubilizing capabilities. Light microscopy, biochemical tests and 16S rRNA gene sequencing revealed that strains 15a1 and 40a were closely related to Enterobacter hormaechei while strain 38 was closely related to Enterobacter cloacae (Accession number: MN294583; MN294585; MN294584). All Enterobacter spp. shared common plant growth-promoting traits, namely nitrogen (N2) fixation, indole-3-acetic acid production and siderophore production. The strains 38 and 40a were able to produce gibberellic acid, while only strain 38 was able to secrete exopolysaccharide on agar. Under in vitro germination assay of okra (Abelmoschus esculentus) seeds, Enterobacter spp. significantly improved overall germination parameters and vigor index (19.6%) of seedlings. The efficacy of root colonization of Enterobacter spp. on the pre-treated seedling root tips was confirmed using Scanning Electron Microscopy (SEM). The pot experiment of bioprimed seeds of okra seedling showed significant improvement of the plant growth (> 28%) which corresponded to the increase of P and K uptakes (> 89%) as compared to the uninoculated control plants. The leaf surface area and the SPAD chlorophyll index of bioprimed plants were increased by up to 29% and 9% respectively. This report revealed that the under-explored species of P- and K-solubilizing Enterobacter hormaechei sp. with multiple plant beneficial traits presents a great potential sustainable approach for enhancement of soil fertility and P and K uptakes of plants.

Cochliobolus sp. acts as a biochemical modulator to alleviate salinity stress in okra plants.

Salinity stress can severely affect the growth and production of the crop plants. Cheap and reliable actions are needed to enable the crop plants to grow normal under saline conditions. Modification at the molecular level to produce resistant cultivars is one of the promising, yet highly expensive techniques, whereas application of endophytes on the other hand are very cheap. In this regard, the role of Cochliobolus sp. in alleviating NaCl-induced stress in okra has been investigated. The growth and biomass yield, relative water content, chlorophyll content and IAA were decreased, whereas malondialdehyde (MDA) and proline content were increased in okra plants treated with 100 mM NaCl. On the contrary, okra plants inoculated with C. lunatus had higher shoot length, root length, plant dry weight, chlorophyll, carotenoids, xanthophyll, phenolicss, flavonoids, IAA, total soluble sugar and relative water content, while lower MDA. LC-MS/MS analysis of the Cochliobolus sp. extract revealed the presence of pinocembrin, chlorogenic acids, wogonin, calycosin and diadzein as a salinity stress reliever. From the results, it can be concluded that colonization of Cochliobolus sp. improves the NaCl tolerance by ameliorating the physicochemical attributes of the host plants.

Assessment of Plant-Probiotic Performance of Novel Endophytic Bacillus sp. in Talc-Based Formulation.

Endophytic bacteria are considered to have a plethora of plant growth promoting and anti-phytopathogenic traits to live within the plants. Hence, they have immense promises for plant probiotic development. In the current study, plant probiotic endophytic Bacillus sp. CaB5 which has been previously isolated from Capsicum annuum was investigated for its performance in talc-based formulation. For this, CaB5 was made into formulation with sterile talc, calcium carbonate, and carboxymethyl cellulose. The viability analysis of the formulation by standard plate count and fluorescence methods has confirmed the stable microbial count up to 45 days. Plant probiotic performance of the prepared formulation was analyzed on cowpea (Vigna unguiculata) and lady's finger (Abelmoschus esculentus). The results showed the formulation treatment to have enhancement effect on seed germination as well as plant growth in both selected plants. The results highlight the potential of CaB5-based formulation for field application to enhance growth of economically important plants.

Biochemical and histochemical analyses revealing endophytic Alcaligenes faecalis mediated suppression of oxidative stress in Abelmoschus esculentus challenged with Sclerotium rolfsii.

Sclerotium rolfsii is a highly aggressive pathogen that causes huge economic losses, especially in temperate climates. Alcaligenes faecalis, particularly in endophytic form, has rarely been used to control this fungus. In this study, endophytic Alcaligenes sp. strain BHU 12, BHU 16 (isolated from Abelmoschus esculentus leaf) and BHU M7 (isolated from Andrographis paniculata leaf) were reported to trigger a wide range of host defenses in Okra plant against the collar-rot pathogen S. rolfsii. Endophytic colonization of the strains in ten days old plants was assessed through re-isolation of the rif-tagged strains on rifampicin augmented nutrient agar media. The ability of the endophytic strains to induce systemic defense responses in above-ground organs was assessed by collecting leaf tissues of the Okra plants grown under non-gnotobiotic conditions at different time intervals post seedling bacterization with the endophytic biocontrol agents. The pathogen challenged unprimed plants exhibited flaccidity of the stem and leaves at 48 h post infection (hpi) in contrast to the bioprimed and challenged plants. Biochemical and histochemical analyses explained the above phenomenon as activation of phyto-peroxidases leading to an increased metabolism of the reactive oxygen species (ROS), accompanied by activation of the phenylpropanoid network and a subsequent enhancement in plant phenolics. Interestingly, though the maximum increase in the defense pathways was observed in treatments with native endophytes of Okra plant, yet the enhancement in antioxidant pathway due to A. paniculata borne endophytes was also quite significant. Thus, this work clearly demonstrates how Okra plants respond to the "non-hostile" colonization of bacterial endophytes and how induced defense response can contribute to the biocontrol activity of the endophytic strains.

Clonal Expansion and Migration of a Highly Virulent, Defoliating Lineage of Verticillium dahliae.

We used a population genomics approach to test the hypothesis of clonal expansion of a highly fit genotype in populations of Verticillium dahliae. This fungal pathogen has a broad host range and can be dispersed in contaminated seed or other plant material. It has a highly clonal population structure, with several lineages having nearly worldwide distributions in agricultural crops. Isolates in lineage 1A are highly virulent and cause defoliation in cotton, okra, and olive (denoted 1A/D), whereas those in other lineages cause wilting but not defoliation (ND). We tested whether the highly virulent lineage 1A/D could have spread from the southwestern United States to the Mediterranean basin, as predicted from historical records. We found 187 single-nucleotide polymorphisms (SNPs), determined by genotyping by sequencing, among 91 isolates of lineage 1A/D and 5 isolates in the closely related lineage 1B/ND. Neighbor-joining and maximum-likelihood analyses on the 187 SNPs showed a clear divergence between 1A/D and 1B/ND haplotypes. Data for only 77 SNPs were obtained for all 96 isolates (no missing data); lineages 1A/D and 1B/ND differed by 27 of these 77 SNPs, confirming a clear divergence between the two lineages. No evidence of recombination was detected within or between these two lineages. Phylogenetic and genealogical analyses resulted in five distinct subclades of 1A/D isolates that correlated closely with geographic origins in the Mediterranean basin, consistent with the hypothesis that the D pathotype was introduced at least five times in independent founder events into this region from a relatively diverse source population. The inferred ancestral haplotype was found in two isolates sampled before 1983 from the southwestern United States, which is consistent with historical records that 1A/D originated in North America. The five subclades coalesce with the ancestral haplotype at the same time, consistent with a hypothesis of rapid population expansion in the source population during the emergence of 1A/D as a severe pathogen of cotton in the United States.

Plant Growth-Promoting Rhizobacteria Enhance Salinity Stress Tolerance in Okra through ROS-Scavenging Enzymes.

Salinity is a major environmental stress that limits crop production worldwide. In this study, we characterized plant growth-promoting rhizobacteria (PGPR) containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and examined their effect on salinity stress tolerance in okra through the induction of ROS-scavenging enzyme activity. PGPR inoculated okra plants exhibited higher germination percentage, growth parameters, and chlorophyll content than control plants. Increased antioxidant enzyme activities (SOD, APX, and CAT) and upregulation of ROS pathway genes (CAT, APX, GR, and DHAR) were observed in PGPR inoculated okra plants under salinity stress. With some exceptions, inoculation with Enterobacter sp. UPMR18 had a significant influence on all tested parameters under salt stress, as compared to other treatments. Thus, the ACC deaminase-containing PGPR isolate Enterobacter sp. UPMR18 could be an effective bioresource for enhancing salt tolerance and growth of okra plants under salinity stress.

Role of Antagonistic Microorganisms and Organic Amendment in Stimulating the Defense System of Okra Against Root Rotting Fungi.

Without application of chemical pesticides control of soilborne diseases is a great challenge. Stimulation of natural plant's defense is considered as one of the most promising alternative strategy for crop protection. Organic amendment of soil besides direct suppressing the pathogen, has been reported to have an influence on phytochemicals in plants. In the present study, Pseudomonas aeruginosa, a plant growth promoting rhizobacterium and Paecilomyces lilacinus, an egg parasite of root knot and cysts nematodes were examined individually and in combination in soil amended with cotton cake for suppressing the root rotting fungi and stimulating the synthesis of polyphenols and improving the antioxidant status in okra. Application of P. aeruginosa and P. lilacinus in soil amended with cotton cake significantly (P < 0.05) suppressed Macrophomina phaseolina, Fusarium oxysporum, and Fusarium solani with complete reduction of Rhizoctonia solani. Combine use of biocontrol agents in cotton cake amended soil showed maximum positive impact on plant growth, polyphenol concentration and antioxidant activity in okra.