Dual Plate Assay Research Articles

Culture-dependent analysis of seed bacterial endophyte, Pseudomonas spp. EGN 1 against the stem rot disease (Sclerotium rolfsii Sacc.) in groundnut

BackgroundGroundnut, Arachis hypogaea L., crop is an important oil seed crop in India and is prone to attack by numerous fungal and viral diseases, among the soil-borne diseases, stem rot caused by Sclerotium rolfsii Sacc. is economically important.Main body of abstractIncidence (86.6%) of the stem rot disease was recorded in Coimbatore district, Tamil Nadu, India, during the roving survey. For its management, an attempt was made to isolate bacterial endophyte from peanut seeds of different groundnut cultivars by culture-dependent analysis. Totally, 16 bacterial endophytes (endophytes groundnut (EGN) 1 to EGN 16 along with standards TNAU-Pf1 and EPC 5) were obtained and characterized through morphological, biochemical and molecular studies and also phytostimulation activities were performed. Among the isolates, EGN 1 and EGN 4 showed positive results for indole acetic acid (IAA), siderophore, phosphate solubilization and protease tests in vitro. The dual culture analysis showed inhibition rates of 60.1% (dual plate assay), 68.23% (filter paper disc assay) and 100% (triangle method of streaking and culture filtrate assay) for EGN 1 against S. rolfsii. Further, the crude metabolite assay showed 97.7% inhibition in EGN 1, followed by 87.7% in EGN 4. The roll towel study showed a high vigour index of 4286.7 in EGN 1; hence, this isolate was chosen. Further, thin-layer chromatography (TLC) analysis showed various bands at 0.72 Rf, whereas GC-MS analysis indicated the prominent peaks of hexadecanoic acid and cis-vaccenic acid that may responsible for antifungal activity. In a molecular approach, the genomic DNA of EGN 1 strain was used to amplify a 1200 bp PCR-fragment and sequenced.Short conclusionThe overall outcome of this study showed that Pseudomonas spp. EGN 1 had a great potential as a bio-stimulant and biocontrol agent to manage effectively the stem rot in peanut.

Trichoderma Isolates Inhibit Fusarium virguliforme Growth, Reduce Root Rot, and Induce Defense-Related Genes on Soybean Seedlings.

Sudden death syndrome (SDS) caused by Fusarium virguliforme is among the most important diseases affecting soybean in the United States. The use of biological control agents (BCAs) such as Trichoderma spp. can be a valuable resource to suppress F. virguliforme populations. Therefore, this research focused on screening possible BCAs against F. virguliforme and evaluating mycoparasitism and the induction of systemic resistance as mechanisms underlying the antagonistic activity of selected BCAs against F. virguliforme. In total, 47 potential BCAs, including 41 Trichoderma isolates and 6 Mortierella isolates, were screened in a dual-plate assay. The most effective isolates belonged to the Trichoderma harzianum species and were able to inhibit F. virguliforme radial growth by up to 92%. Selected Trichoderma isolates were tested in the greenhouse and in a microplot study. They reduced root rot caused by F. virguliforme when the plants were coinoculated with the pathogen and the BCA. The tested BCA's ability to reduce F. virguliforme growth may be related to several mechanisms of action, including mycoparasitism and induction of defense-related genes in plants, as revealed by monitoring the expression of defense-related genes in soybean. Our results highlight the potential of native Trichoderma isolates to inhibit F. virguliforme growth and reduce SDS severity, providing the basis for future implementation of biological control in soybean production. More efforts are needed to implement the use of these approaches in production fields, and to deepen the current knowledge on the biology of these highly antagonistic isolates.

Effect of the endophytic plant growth promoting Enterobacter ludwigii EB4B on tomato growth

Summary This study aims to develop a biocontrol agent against Fusarium oxysporum f.sp. radicis-lycopersici (FORL) in tomato. For this, a set of 23 bacterial endophytic isolates has been screened for their ability to inhibit in vitro the growth of FORL using the dual plate assay. Three isolates with the most sound antagonistic activity to FORL have been qualitatively screened for siderophore production, phosphates solubilization and indolic acetic acid (IAA) synthesis as growth promotion traits. Antagonistic values of the three candidates against FORL were respectively: 51.51 % (EB4B), 51.18 % (EB22K) and 41.40 % (EB2A). Based on 16S rRNA gene sequence analysis, the isolates EB4B and EB22K were closely related to Enterobacter ludwigii EN-119, while the strain EB2A has been assigned to Leclercia adecarboxylata NBRC 102595. The promotion of tomato growth has been assessed in vitro using the strains EB2A, EB4B and EB22K in presence of the phytopathogen FORL. The treatments with the selected isolates increased significantly the root length and dry weight. Best results were observed in isolate EB4B in terms of growth promotion in the absence of FORL, improving 326.60 % of the root length and 142.70 % of plant dry weight if compared with untreated controls. In the presence of FORL, the strain EB4B improved both root length (180.81 %) and plant dry weight (202.15 %). These results encourage further characterization of the observed beneficial effect of Enterobacter sp. EB4B for a possible use as biofertilizer and biocontrol agent against FORL.

Seed Biopriming with Microbial Inoculant Triggers Local and Systemic Defense Responses against Rhizoctonia solani Causing Banded Leaf and Sheath Blight in Maize (Zea mays L.).

Plant growth promoting rhizobacteria Pseudomonas aeruginosa strain MF-30 isolated from maize rhizosphere was characterized for several plant growth stimulating attributes. The strain MF-30 was also evaluated for antifungal properties against Rhizoctonia solani causing banded leaf and sheath blight in maize (Zea mays L.) under in vitro conditions and was found to have higher mycelial growth suppression in the culture suspension (67.41%) followed by volatile organic compounds (62.66%) and crude extract (51.20%) in a dual plate assay. The endophytic and epiphytic colonization ability was tested using Green Fluorescent Protein (GFP)-tagging. Visualization through confocal scanning laser microscope clearly indicated that strain MF-30 colonizes the root and foliar parts of the plants. Further, the effects of seed bio-priming with P. aeruginosa MF-30 was evaluated in the induction and bioaccumulation of defense-related biomolecules, enzymes, natural antioxidants, and other changes in maize under pot trial. This not only provided protection from R. solani but also ensured growth promotion under pathogenic stress conditions in maize. The maximum concentration of hydrogen peroxide (H2O2) was reported in the root and shoot of the plants treated with R. solani alone (8.47 and 17.50 mmol mg−1 protein, respectively) compared to bioagent, P. aeruginosa MF-30 bio-primed plants (3.49 and 7.50 mmol mg−1 protein, respectively). Effects on total soluble sugar content, total protein, and total proline were also found to enhanced significantly due to inoculation of P. aeruginosa MF-30. The activities of anti-oxidative defense enzymes phenylalanine ammonia lyase (PAL), ascorbate peroxidase, peroxidase, superoxide dismutase, and catalase increased significantly in the plants bio-primed with P. aeruginosa MF-30 and subsequent foliar spray of culture suspension of MF-30 compared to pathogen alone inoculated plants. qRT-PCR analysis revealed that seed bio-priming and foliar application of P. aeruginosa MF-30 significantly increased the expression of PR-1 and PR-10 genes with the simultaneous decrease in the disease severity and lesion length in the maize plants under pathogenic stress conditions. A significant enhancement of shoot and root biomass was recorded in MF-30 bio-primed plants as compared to untreated control (p < 0.05). Significant increase in plant growth and antioxidant content, as well as decreased disease severity in the P. aeruginosa MF-30 bio-primed plants, suggested the possibility of an eco-friendly and economical means of achieving antioxidants-rich, healthier maize plants.

Antifungical Activity of Autochthonous Bacillus subtilis Isolated from Prosopis juliflora against Phytopathogenic Fungi

The ability of Bacillus subtilis, strain ALICA to produce three mycolytic enzymes (chitinase, β-1,3-glucanase, and protease), was carried out by the chemical standard methods. Bacillus subtilis ALICA was screened based on their antifungal activity in dual plate assay and cell-free culture filtrate (25%) against five different phytopathogenic fungi Alternaria alternata, Macrophomina sp., Colletotrichum gloeosporioides, Botrytis cinerea, and Sclerotium rolfesii. The B. subtilis ALICA detected positive for chitinase, β-1,3-glucanase and protease enzymes. Fungal growth inhibition by both strain ALICA and its cell-free culture filtrate ranged from 51.36% to 86.3% and 38.43% to 68.6%, respectively. Moreover, hyphal morphological changes like damage, broken, swelling, distortions abnormal morphology were observed. Genes expression of protease, β-1,3-glucanase, and lipopeptides (subtilosin and subtilisin) were confirmed their presence in the supernatant of strain ALICA. Our findings indicated that strain ALICA provided a broad spectrum of antifungal activities against various phytopathogenic fungi and may be a potential effective alternative to chemical fungicides.

Antagonistic assessment of Trichoderma spp. by producing volatile and non-volatile compounds against different fungal pathogens

Trichoderma spp. are well-known biological agents that have significant antagonistic activity against several plant pathogenic fungi. In the present study, Trichoderma spp. were tested in vitro for their antagonistic activity against different spp. of Fusarium and Alternaria viz. Alternaria alternata, A. brassicae, A. solani, Fusarium oxysporum and F. solani using dual plate assay and by the production of volatile and non-volatile compounds. The results obtained revealed that Trichoderma harzianum and T. viride effectively inhibited the growth and spore production of different spp. of Fusarium and Alternaria. The highest growth inhibition was found in A. alternata 62.50% and 60.00% by non-volatile compounds of T. harzianum and T. viride, respectively. Similarly, the volatile compounds inhibit the maximum growth of A. alternata 40.00% and 35.00% by T. harzianum and T. viride, respectively. Volatile and non-volatile compounds of Trichoderma spp. were analysed by GC-MS technique and the properties of distinguished compounds showed antifungal, antimicrobial and antibiotic activities. Volatile compounds of T. harzianum and T. viride showed highest percent abundance for glacial acetic acid (45.32%) and propyl-benzene (41.75%), respectively. In case of non-volatile compounds, T. harzianum and T. viride showed D-Glucose, 6-O-α-D-galactopyranosyl- (38.45%) and 17-Octadecynoic acid (36.23%), respectively. The results of present study confirmed that T. harzianum can be used as a promising biological control agent against Alternaria and Fusarium spp. that cause diseases in various vegetables and crops.

TWO TRICHODERMA SPECIES AND BACILLUS SUBTILIS AS BIOCONTROL AGENTS AGAINST RHIZOCTONIA DISEASE AND THEIR INFLUENCE ON POTATO PRODUCTIVITY

Stem canker and black scurf caused by Rhizoctonia solani is a problem facing potato production. In this work, under greenhouse conditions, three compatible bioagents i.e., Trichoderma koningii and T. harzianum (in mixture) and Bacillus subtilis ATCC®11774™ were evaluated individually and in combinations for disease suppression and further effect on plant growth of potato plants. Radial growth of R. solani was inhibited by the two Trichoderma strains and B. subtilis in dual Petri plate assay. In two experiments, significant plant protection was achieved when either B. subtilis added to tubers or Trichoderma mixture was added to the soil. However, soil application with Trichoderma either singly or in combination with tuber bacterization demonstrated the greatest suppression of cankers on potato plants. With respect to plant growth promotion, the greatest proportional increases in plant height were elicited by tuber bacterization combined with soil application of Trichoderma mixture. Dual tuber treatments by Trichoderma mixture with soil applications of bacteria led to the highest increase of plant stolons and leaf numbers in both experiments. Both combined applications and sole soil application by T. mixture recorded the same significant effect in increasing shoot fresh and dry weights of potato plants as well as improved tuber yield and some biochemical parameters (chlorophyll content, total phenol, peroxidase and polyphenoloxidase contents) significantly. This research suggests incorporation of such bioagents to suppress Rhizoctonia diseases and increase the productivity of potato. S

Antifungal activity of indigenous Bacillus spp. isolated from soil

Biocontrol using plant growth-promoting rhizobacteria (PGPR) represents an alternative approach to disease management, since PGPR are known to promote growth and reduce diseases in various crops. Among the different PGPR, members of the genus Bacillus are prefered for most biotechnological uses due to their capability to form extremely resistant spores and produce a wide variety of metabolites with antimicrobial activity. The objective of this research was to identify antagonistic bacteria for management of the plant diseases. Eleven isolates of Bacillus spp. were obtained from the soil samples collected from different localities in the Province of Vojvodina. The antifungal activity of bacterial isolates against five fungal species was examined using a dual plate assay. Bacillus isolates exhibited the highest antifungal activity against Fusarium proliferatum, Fusarium oxysporum f. sp. cepae and Alternaria padwickii, while they had the least antagonistic effect on Fusarium verticillioides and Fusarium graminearum. Molecular identification showed that effective bacterial isolates were identified as Bacillus safensis (B2), Bacillus pumilus (B3, B11), Bacillus subtilis (B5, B7) and Bacillus megaterium (B8, B9). The highest antagonistic activity was exhibited by isolates B5 (from 39% to 62% reduction in fungal growth) and B7 (from 40% to 71% reduction in fungal growth). These isolates of B. subtilis could be used as potential biocontrol agents of plant diseases.

Bio-protective microbial agents from rhizosphere eco-systems trigger plant defense responses provide protection against sheath blight disease in rice (Oryza sativa L.)

Sheath blight of rice (Oryza sativa L.) caused by Rhizoctonia solani is a major disease and attempts are being made to develop microbe based technologies for biocontrol of this pathogen. However, the mechanisms of biocontrol are not fully understood and still require indepth study in the backdrop of emerging concepts in biological systems. The present investigation was aimed at deciphering the mechanisms of biocontrol of sheath blight of rice employing Pseudomonas fluorescens and Trichoderma harzianum as model agents for biocontrol. Initially 25, 5 and 5 strains of P. fluorescens, T. viride and T. harzianum, respectively, were screened for their biocontrol potential. Out of which, six strains with higher value of percent inhibition of fungal mycelium in dual plate assay were selected. The role of P. fluorescens, T. viride and T. harzianum were investigated in induction and bioaccumulation of natural antioxidants, defence-related biomolecules and other changes in plant which lead not only to growth promotion but also protection from pathogenic stress conditions in rice. The two most promising strains, P. fluorescens PF-08 and T. harzianum UBSTH-501 selected on the basis of in planta evaluation, when applied individually or in combination, significantly enhanced the accumulation of defence-related biomolecules, enzymes and exhibited biocontrol potential against R. solani. A modified/newly developed delivery system was applied for the first time in the experiments involving inoculation of plants with both bioagents, viz. P. fluorescens PF-08 and T. harzianum UBSTH-501. Results suggested that application of P. fluorescens PF-08 and T. harzianum UBSTH-501 alone or in combination, not only helps in control of the disease but also increases plant growth along with reduction in application of toxic chemical pesticides.

Biosynthesis and characterization of a thermostable, alkali-tolerant chitinase from Bacillus pumilus JUBCH08 displaying antagonism against phytopathogenic Fusarium oxysporum.

The present investigation highlights the process parameters influencing the submerged fermentation of chitinase by Bacillus pumilus JUBCH08, purification and characterization of the enzyme and determination of antagonistic activity of the bacterium against Fusarium oxysporum. Medium supplemented with 0.5 % chitin and peptone, at initial pH 8.0, when incubated at 35 °C for 72 h favored highest chitinase production. The enzyme was purified 25.1-fold to homogeneity. The chitinase was found to be thermostable and alkali-tolerant with maximum activity at pH 8.0 and 70 °C for 1 h. The molecular weight of chitinase was found to be 64 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Mg2+, Co2+, Ca2+ and Mn2+ improved the chitinase activity. The K m and V max values of the enzyme were 0.13 mg/ml and 38.23 U/ml, respectively. When subjected to dual plate assay, the bacterium showed 45 % antagonism against F. oxysporum. Thus, it could be inferred that cultural conditions strongly affected the chitinase production by B. pumilus JUBCH08. The enzyme being thermostable and best functional under alkaline conditions could be useful for the feed industry and related biotechnological applications. Inhibition of F. oxysporum by the culture through lytic mechanism indicates its potentiality as a biocontrol agent.

Biocontrol of &amp;lt;i&amp;gt;Rhizoctonia solani&amp;lt;/i&amp;gt; K1 by Iturin A Producer &amp;lt;i&amp;gt;Bacillus subtilis&amp;lt;/i&amp;gt; RB14 Seed Treatment in Tomato Plants

Bacillus subtilis RB14 was used as an antagonist against fungal pathogen Rhizoctonia solani K1 to control damping-off diseases in tomato plants. Tomato seeds were treated with B. subtilis RB14 culture. The concentration of bacterial cells for the treatment was about 108 cfu/ml. Treated tomato seeds showed 99% germination index similar to the untreated seeds. Scanning Electron Microscopic observations showed a clear evidence of the presence of B. subtilis RB14 on tomato seed surface. Clear inhibition zone was observed using treated seed in dual plate assay against R. solani K1. B. subtilis RB14 treated seed showed 80% reduction in disease incidence during in vivo plant experiments. B. subtilis RB14 produces lipopeptide antifungal antibiotic iturin A which could suppress R. solani K1. The phenomenon was supported by our observation where we found significant amount of iturin A from the root zone soil of the seed treated plants.

The ABC transporter ABCG29 is involved in H2O2 tolerance and biocontrol traits in the fungus Clonostachys rosea.

For successful biocontrol interactions, biological control organisms must tolerate toxic metabolites produced by themselves or plant pathogens during mycoparasitic/antagonistic interactions, by host plant during colonization of the plant, and xenobiotics present in the environment. ATP-binding cassette (ABC) transporters can play a significant role in tolerance of toxic compounds by mediating active transport across the cellular membrane. This paper reports on functional characterization of an ABC transporter ABCG29 in the biocontrol fungus Clonostachys rosea strain IK726. Gene expression analysis showed induced expression of abcG29 during exposure to the Fusarium spp. mycotoxin zearalenone (ZEA) and the fungicides Cantus, Chipco Green and Apron. Expression of abcG29 in C. rosea was significantly higher during C. rosea-C. rosea (Cr-Cr) interaction or in exposure to C. rosea culture filtrate for 2 h, compared to interaction with Fusarium graminearum or 2 h exposure to F. graminearum culture filtrate. In contrast with gene expression data, ΔabcG29 strains did not display reduced tolerance towards ZEA, fungicides or chemical agents known for inducing oxidative, cell wall or osmotic stress, compared to C. rosea WT. The exception was a significant reduction in tolerance to H2O2 (10 mM) in ΔabcG29 strains when conidia were used as an inoculum. The antagonistic ability of ΔabcG29 strains towards F. graminearum, Fusarium oxysporum or Botrytis cinerea in dual plate assays were not different compared with WT. However, in biocontrol assays ΔabcG29 strains displayed reduced ability to protect Arabidopsis thaliana leaves from B. cinerea, and barley seedling from F. graminearum as measured by an A. thaliana detached leaf assay and a barley foot rot disease assay, respectively. These data show that the ABCG29 is dispensable for ZEA and fungicides tolerance, and antagonism but not H2O2 tolerance and biocontrol effects in C. rosea.

Endophytes from wheat as biocontrol agents against tan spot disease

Endophytes from wheat cultivars isolated in Buenos Aires province, Argentina, were assessed for their potential as biocontrol agents against Pyrenophora tritici-repentis (Died.) Drechsler (anamorph Drechslera tritici-repentis) (Died.) Shoem (Dtr), the causal agent of tan spot of wheat. Endophytes were screened using dual culture techniques and examining the effect on growth, sporulation and the antifungal activity in greenhouse assays. The most of endophytes tested significantly reduced Dtr growth compared to the control except Rhodotorula rubra from 11 to 15days post inoculation. Trichoderma hamatum, Penicillium sp., Bacillus sp. and Paecilomyces lilacinus significantly reduced the colony diameter of the pathogen. Most of the endophytes evaluated showed morphological changes in the conidia and/or the mycelia of D. tritici-repentis. In addition, two endophytes, Bacillus sp. and Fusarium sp., reduced significantly the percent spore germination of Dtr compared to the control by 82% and 52% respectively. In greenhouse experiment T. hamatum, Chaetomium globosum and Fusarium sp. significantly (P⩽0.05) reduced the average disease severity on all three leaves compared to the control. However, the best antagonistic effect was shown with T. hamatum as it resulted in the greatest suppression in the greenhouse and in the dual-plate assays. Likewise, Bacillus sp. was other highlighted microorganism that antagonized the pathogen in in vitro assays. From our promising results, we conclude that endophytes have potential in the biological control of tan spot of wheat caused by D. tritici-repentis, particularly T. hamatum and Bacillus sp.

Trichoderma from Aceh Sumatra reduce Phytophthora lesions on pods and cacao seedlings

The cocoa tree, Theobroma cacao L., suffers large yield losses in Aceh Indonesia due to the disease black pod rot, caused by Phytophthora spp. Despite having the largest area under cacao production in Sumatra, farmers in the Aceh region have low overall production because of losses to insect pests and black pod rot. Trichoderma spp. were isolated from the roots and leaves of cacao trees and screened as potential biological control agents. Isolates used in the study were Trichoderma asperellum isolates T2 and T4, Trichoderma longibrachiatum isolates T15 and T16, and Trichoderma virens isolates T1 and Tv. T1, T2, T4, and Tv completely colonized and destroyed Phytophthora tropicalis and Phytophthora palmivora mycelium in precolonized plate assays. All six isolates reduced P. tropicalis, but none reduced the growth of P. palmivora in dual plate assays. Phytophthora growth was suppressed on MIN media amended with sterile heat inactivated Trichoderma culture filtrates, with Tv best suppressing growth of both Phytophthora spp. T. virens isolate Tv was the only isolate observed coiling around P. tropicalis mycelium and disrupted the formation of P. palmivora sporangia. Of all six isolates, only Tv reduced P. palmivora lesion expansion in a detached pod assay, reducing severity by 71%. Tv also reduced P. palmivora infection on seedlings when applied aerially at 1×106 and 1×108conidia/ml, by 19% and 59%, respectively. T. virens isolate Tv is a mycoparasite, antagonizes Phytophthora in a dual plate assay, and shows antibiosis against Phytophthora spp., suggesting that multiple modes of action contribute to its ability to limit Phytophthora lesion expansion on cacao pods and seedlings.

Additive and synergistic effects of Bacillus spp. isolates and essential oils on the control of phytopathogenic and saprophytic fungi from medicinal plants and marigold seeds

Contamination of plants and seeds with microorganisms is one of the main problems in the production and distribution of various agricultural products, as well as raw herbal material for the preparation of herbal remedies. In targeting microbial contamination, among other bacteria, Bacillus species showed a significant capacity for biocontrol. The antifungal activity of 14 isolates of Bacillus spp. against 15 fungal isolates from medicinal plants was examined utilizing a dual plate assay. The strongest and broadest antagonistic activity against all fungi tested was exhibited by isolates SS-12.6 and SS-13.1 (from a 43% to 74% reduction in fungal growth), while isolates SS-39.1 and SS-39.3 were effective against the fewest fungus species and also had the weakest antifungal activity. The effect of a crude lipopeptide extract (CLE) of Bacillus sp. SS-12.6 was similar to that achieved by a dual culture with isolate SS-12.6, confirming that the antagonism was the result of the antifungal activities of lipopeptides. In addition, essential oils of thyme (0.55mg/mL) and savory (0.32mg/mL) in various combinations with the CLE of SS-12.6 were tested for antifungal activity, and additive and synergistic effects for some of the fungi were obtained. When testing the effect of CLE, oils (0.40mg/mL for thyme oil and 0.21mg/mL for savory oil) and combinations in situ on marigold seeds, a reduction of total fungal infection without an adverse effect on germination was accomplished by 6-h treatments with CLE of SS-12.6 (85% reduction of fungal infection and 63% germination), supernatant from liquid culture of SS-12.6 (more than 90% reduction of fungal infection with 69% seed germination) and combinations of CLE and savory oil (77% reduction of fungal infection and 62% seed germination) and CLE with thyme and savory oils (about 75% reduction of fungal infection with 69% seed germination).

Rhizosphere Bacteria for Biocontrol of Bacterial Blight and Growth Promotion of Rice

Several bacterial strains were isolated from different rhizospheres. Among these, strain PDY7 exhibited strong antibacterial activity against the rice bacterial blight (BB) pathogen Xanthomonas oryzae pv. oryzae (Xoo) by the laboratory dual plate assays. The antibacterial property of the strain PDY7 was further investigated for the production of 2,4-diacetylphloroglucinol (DAPG), which amplified a characteristic of 629-bp DNA fragment by PCR-based screening method using phlD primers. The application of phlD positive strains was carefully evaluated for disease control and growth promotion of rice plants under field conditions. The selected strain PDY7 suppressed the rice BB by 58.83% and 51.88% under glass house and field conditions, respectively. In addition, the strain PDY7 showed significant two-fold increase in root length (18.08 cm), shoot length (29.81 cm), and grain yield (96.07 g). Strain PDY7 promoted the growth of rice plants by production of indole-3-acetic acid (IAA), which was determined by high performance liquid chromatography (HPLC) analysis. Our findings suggest that PDY7 belongs to the P. fluorescens group and can serve as potential biocontrol of BB as well as biofertilizer agent for growth promotion of rice.

Rhizobacterium-mediated growth promotion and expression of stress enzymes in Glycine max L. Merrill against Fusarium wilt upon challenge inoculation

Wilt disease of soybean caused by a very common soil-borne fungus, Fusarium oxysporum is one of the most destructive diseases of the crop. The aim of the present study was to characterize plant growth-promotion activities and induced resistance of a rhizobacterial strain for the soybean plant against F. oxysporum. Rhizobacterium strain SJ-5 exhibited plant growth-promotion characteristics and antagonistic activity against the test pathogen on dual plate assay. It was identified as a Carnobacterium sp. A 950 bp PCR product was amplified from Carnobacterium sp. strain SJ-5, using zwittermicin A self-resistance gene-specific primers (zmaR). The strain produced indole 3-acetic acid (19 μg/ml) in the presence of salt stress and exhibited growth in Dworkin and Foster salt medium amended with 1-aminocyclopropane-1-carboxylate (ACC) through ACC deaminase activity (277 nmol/mg/h) as compared to the control. Strain seeds treated with the strain significantly enhanced the quorum of healthy plants after challenge inoculation at 14 days after seeding. An increase in the activity of stress enzymes after challenge inoculation with the test pathogen is reported. Treatment with the bacterium resulted in an increase in the chlorophyll content in the leaves in comparison with challenge-inoculated plants.

Potentiality of Bacillus subtilis as biocontrol agent for management of anthracnose disease of chilli caused by Colletotrichum gloeosporioides OGC1.

A soil bacterium, Bacillus subtilis, isolated from the rhizosphere of Chilli, showed high antagonistic activity against Colletotrichum gloeosporioides OGC1. A clear inhibition zone of 0.5–1 cm was observed in dual plate assay. Microscopic observations showed a clear hyphal lysis and degradation of fungal cell wall. In dual liquid cultures, the B. subtilis strain inhibited the C. gloeosporioides up to 100 % in terms of dry weight. This strain also produced a clear halo region on chitin agar medium plates containing 0.5 % colloidal chitin, indicating that it excretes chitinase. The strain also produced other mycolytic enzymes—glucanase and cellulase, demonstrated by a clear zone of hydrolysis of yeast cell wall glucan (YCW 0.1 % v/v) and carboxymethylcellulose (CMC 0.1 % v/v). In liquid cultures, the strain showed appreciable levels of chitinase, glucanase and cellulase activities and hydrolytic activity with C. gloeosporioides OGC1 mycelia as the substrate. The role of the B. subtilis strain in suppressing the fungal growth in vitro was studied in comparison with a UV mutant of that strain, which lacked both antagonistic and hydrolytic activity. The mycolytic enzyme mediated antagonism of B. subtilis was further demonstrated by heat inactivation (70–100 °C), treatment with trypsin and TCA of the crude enzyme extract which lacked antifungal property also. Treatment of the chilli seeds with Bacillus sp. culture showed 100 % germination index similar to the untreated seeds. The treatment of the seed with co-inoculation of the pathogen with Bacillus sp. culture showed 65 % reduction in disease incidence by the treatment as compared to the seed treated with pathogen alone (77.5 %).

Enhancement of mycolytic activity of an antagonistic Bacillus subtilis through ethyl methane sulfonate (EMS) mutagenesis

Bacillus subtilis, isolated from the chili rhizosphere, capable of producing 3 mycolytic enzymes (chitinase, \beta-1,3-glucanase, and cellulase), was mutated by the chemical mutagen ethyl methane sulfonate (EMS). Mutants were screened based on their antifungal ability on dual plate assay against Colletotrichum gloeosporioides OGC1, the causal agent of anthracnose disease in fruit crops. EMS mutagenesis yielded 60 isolated mutants on NA plates, of which 3 (M3, M4, and M24) showed loss of antagonism against C. gloeosporioides OGC1 and 6 (M8, M21, M22, M57, M58, and M59) exhibited increased antagonism. The remaining mutants did not show any difference in their antagonistic property. In liquid culture, the mutants exhibited varied levels of the 3 enzymes. These mutants were studied for their mycolytic enzyme activities under shake flask conditions. Of all these mutants, M57 showed a 36-fold and 4.71-fold increase in \beta -1,3-glucanase and cellulase activities, respectively, with a concomitant 1.95-fold increase in hydrolytic activity, followed by M59, with a 5.68-fold and 1.57-fold increase in \beta -1,3-glucanase and cellulase activities, respectively, and a 2.23-fold increase in hydrolytic activity as compared to the wild type B. subtilis strain. M24 exhibited a complete loss of \beta -1,3-glucanase and a decrease in chitinase, with a concomitant decrease in levels of hydrolytic activity with C. gloeosporioides mycelia as compared to the wild type strain. The study clearly indicated the mycolytic enzyme mediated antagonism of this strain and yielded 2 hyperproducing mutants for \beta -1,3-glucanase and cellulase, 2 important enzymes for various agricultural and industrial uses.

PLANT GROWTH-PROMOTING BACTERIAL ENDOPHYTES FROM SUGARCANE AND THEIR POTENTIAL IN PROMOTING GROWTH OF THE HOST UNDER FIELD CONDITIONS

SUMMARYTen endophytic bacteria were isolated from different sugarcane varieties growing in the Crop Research Centre, Pantnagar on nitrogen-free medium. Plant growth-promoting potential of the isolates was reported in terms of indole acetic acid (IAA) production, phosphorus solubilization, siderophore production and antagonistic action against the pathogenColletotrichum falcatum, which causes red rot disease in sugarcanein vitro. All the isolates were able to produce IAA (4.8–9 μg ml−1); three isolates (H3, H5 and H14) solubilized insoluble phosphorus on Pikovaskaya's agar; two isolates (H10 and H14) showed siderophore production on Chrome-azurol S (CAS) agar and antagonism againstC. falcatumwas exhibited by two isolates (H14 and H15) in a dual plate assay. 16 S rRNA sequencing identified isolates H3 and H12 asPseudomonasspp., and H8, H14 and H15 asBacillusspp. A field experiment on sugarcane was conducted with five plant growth-promoting bacterial endophytesPseudomonasspp. (H3 and H12) andBacillusspp. (H8, H14 and H15) along with standard strains ofGluconacetobacterandAzospirillumspp. Plant height, chlorophyll content, total nitrogen and cane length were significantly higher in almost all inoculated plants compared with the uninoculated control. An increase of 40% in cane yield over the control was obtained after inoculation with isolate H15 (Bacillusspp.). This was statistically on par with the standard endophyteGluconacetobacter diazotrophicus, which resulted in 42% increased cane yield. Identification of new diazotrophs and their promising results towards improving plant growth in the field suggest their use as inoculants in future.