Plant Assays Research Articles

HIGH CROSSOVER RATE1 encodes PROTEIN PHOSPHATASE X1 and restricts meiotic crossovers in Arabidopsis.

Meiotic crossovers are tightly restricted in most eukaryotes, despite an excess of initiating DNA double-strand breaks. The majority of plant crossovers are dependent on Class I interfering repair, with a minority formed via the Class II pathway. Class II repair is limited by anti-recombination pathways, however similar pathways repressing Class I crossovers are unknown. We performed a forward genetic screen in Arabidopsis using fluorescent crossover reporters, to identify mutants with increased or decreased recombination frequency. We identified HIGH CROSSOVER RATE1 (HCR1) as repressing crossovers and encoding PROTEIN PHOSPHATASE X1. Genome-wide analysis showed that hcr1 crossovers are increased in the distal chromosome arms. MLH1 foci significantly increase in hcr1 and crossover interference decreases, demonstrating an effect on Class I repair. Consistently, yeast two-hybrid and in planta assays show interaction between HCR1 and Class I proteins, including HEI10, PTD, MSH5, and MLH1. We propose that HCR1 plays a major role in opposition to pro-recombination kinases to restrict crossovers in Arabidopsis.

In planta Activity of Novel Copper(II)-Based Formulations to Inhibit the Esca-Associated Fungus Phaeoacremonium minimum in Grapevine Propagation Material.

Grapevine trunk diseases (GTDs) are a serious and growing threat to vineyards worldwide. The need for innovative control tools persists since pesticides used against some GTDs have been banned and only methods to prevent infections or to reduce foliar symptoms have been developed so far. In this context, the application of imaging methods, already applied to study plant–microbe interactions, represents an interesting approach to understand the effect of experimental treatments applied to reduce fungal colonization, on GTD-related pathogens activity. To this aim, trials were carried out to evaluate the efficacy of copper-based treatments, formulated with hydroxyapatite (HA) as co-adjuvant with innovative delivery properties, loaded with two different copper(II) compounds (tribasic sulfate and sulfate pentahydrate), and applied to grapevine propagation material to inhibit fungal wood colonization. The treated rootstock (Vitis berlandieri × Vitis riparia cv. K5BB) and scion cuttings (Vitis vinifera L., cv. Chardonnay) had been inoculated with a strain of Phaeoacremonium minimum (Pmi) compared to uninoculated rootstocks. Experimental treatments were applied during the water-soaking process, comparing the copper(II) compounds pure or formulated with HA, to hydrate the cuttings. After callusing, grafted vines were grown under greenhouse conditions in a nursery and inoculated with Pmi::gfp7 or with Pmi wild-type. Fifteen weeks post-inoculation, woody tissues close to the inoculation site were sampled to evaluate the efficiency of the treatments by studying the plant–microbe interaction by confocal laser scanning microscopy (CLSM). Copper and further elements were also quantified in the same tissues immediately after the treatments and on the CLSM samples. Finally, the grapevine defense responses were studied in the leaves of cuttings treated with the same formulations. The present investigation confirmed the relevant interaction of Pmi and the related transformed strain on the vascular tissues of grafted vines. Furthermore, in vitro assay revealed (i) the fungistatic effect of HA and the reduced effect of Cu fungicide when combined with HA. In planta assays showed (ii) the reduction of Pmi infection in propagation material treated with HA-Cu formulations, (iii) the movement of HA-Cu formulations inside the plant tissues and their persistence over time, and (iv) the plant defense reaction following the treatment with pure HA or Cu, or combined.

Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)-bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

Saccharin Provides Protection and Activates Defense Mechanisms in Wheat Against the Hemibiotrophic Pathogen Zymoseptoria tritici.

Plant resistance inducers are among the most promising alternatives to develop sustainable crop protection. Here, we examined the ability of saccharin, a metabolite derived from probenazole, to protect wheat against Zymoseptoria tritici, the most frequently occurring and damaging foliar pathogen on this crop. The experiments were performed in the greenhouse by treating seedlings of the wheat cultivar 'Alixan' with 15 mM of saccharin 2 days before challenge inoculation with the Z. tritici pathogenic strain T02596. Foliar application of saccharin resulted in 77% lower disease severity than in nontreated control plants. In vitro and in planta assays showed that saccharin did not exhibit any direct antifungal effect on spore germination or hyphal growth. Molecular investigations from 2 to 7 days posttreatment (dpt) revealed that saccharin treatment upregulates the expression of genes encoding for lipoxygenase (LOX) at all sampled time points and pathogenesis-related protein 1 (PR1) at 7 dpt, in both noninfectious and infectious contexts, as well as peroxidase (POX2) in noninfectious conditions. However, saccharin did not induce significant change in the expression of PAL gene encoding for phenylalanine ammonia-lyase. Our findings report for the first time the potential of saccharin to confer protection in wheat against Z. tritici through an elicitation and priming of LOX and PR gene-related defense pathways. Additional investigations would provide a better deciphering of defense mechanisms activated by this molecule in wheat against Z. tritici.

Characterization of Lysinibacillus fusiformis strain S4C11: In vitro, in planta, and in silico analyses reveal a plant-beneficial microbe

Despite sharing many of the traits that have allowed the genus Bacillus to gain recognition for its agricultural relevance, the genus Lysinibacillus is not as well-known and studied. The present study employs in vitro, in vivo, in planta, and in silico approaches to characterize Lysinibacillus fusiformis strain S4C11, isolated from the roots of an apple tree in northern Italy. The in vitro and in vivo assays demonstrated that strain S4C11 possesses an antifungal activity against different fungal pathogens, and is capable of interfering with the germination of Botrytis cinerea conidia, as well as of inhibiting its growth through the production of volatile organic molecules. In planta assays showed that the strain possesses the ability to promote plant growth, that is not host-specific, both in controlled conditions and in a commercial nursery. Biocontrol assays carried out against phytopathogenic viruses gave contrasting results, suggesting that the strain does not activate the host’s defense pathways. The in silico analyses were carried out by sequencing the genome of the strain through an innovative approach that combines Illumina and High-Definition Mapping methods, allowing the reconstruction of a main chromosome and two plasmids from strain S4C11. The analysis of the genes encoded by the genome contributed to the characterization of the strain, detecting genes related to the biocontrol effect detected in the experimental trials.

Biological control of Fusarium wilt and growth promotion in pigeon pea (Cajanus cajan) by antagonistic rhizobacteria, displaying multiple modes of pathogen inhibition

Fusarium udum is one of the most important pathogens impacting the production of pigeon pea (Cajanus cajan) legume. Legume growers depend heavily on pesticides; however, toxicity danger to farmers, consumers, non-target organisms as well as environmental health cause huge concern. Besides, the rise of fungicide resistance is problematic. The objectives of this study were to decipher the multiple mechanisms of biological control in previously identified two promising rhizobacteria (Pseudomonas sp. NS 1 and Bacillus sp. NS 22) in vitro and evaluate their disease suppression and plant growth promotion ability in pigeon pea in vivo. These rhizobacteria inhibited F. udum mycelia growth and biomass in vitro and reduced wilt disease severity in plants grown in Fusarium-infested soil in controlled environmental conditions. Fungicidal action of these rhizobacteria was due to the production of numerous biocidal compounds including different antifungal metabolites, chitinolytic (endochitinase, exochitinase, chitobiase) and other cell wall degrading lytic enzymes (proteinase, cellulose, amylase, pectinase, lipase), siderophores, and antifungal volatile compounds such as ammonia and cyanide. In planta assay, these rhizobacteria made the plant more resistant to fungal attacks through the induction of systemic defense in host tissue by improving the status of different defense enzymes (phenylalanine ammonia lyase, polyphenol oxidase, and peroxidase) and phenolics. Furthermore, these two bacteria significantly reduced disease severity, root mortality, and at the same time, promoted shoot and root elongation in pigeon pea seedlings as compared to the absolute, pathogen, and chemical control. In nutshell, Pseudomonas sp. NS 1 and Bacillus sp. NS 22 displayed the potential as biofungicides and biofertilizers to prevent F. udum induced disease incidence and promote plant growth in an integrative disease and nutrient management approach of sustainable agriculture practices.

Synergistic effect of oilseed cake and biocontrol agent in the suppression of Fusarium wilt in Solanum lycopersicum.

The antagonistic efficacy of a biocontrol agent in combination with oilseed cake against fungal phytopathogens has been sparsely explored. The present study aimed to evaluate the antifungal activity of a biocontrol agent (Trichoderma harzianum MTCC 3928) formulated with oilseed cake (OSC) against Fusarium oxysporum f. sp. lycopersici responsible for causing vascular wilt in Solanum lycopersicum. In in vitro studies, OSC of mustard (Brassica juncea) exhibited significant mycelial inhibition against the pathogen. The volatile plate assay showed mycelial inhibition of 70 and 40% with unautoclaved and autoclaved mustard cakes, respectively. The aqueous extract (10%v/v) of the mustard cake was the most effective with 47.3% mycelial inhibition of pathogen over control. In addition, volatiles and aqueous extract of mustard cake subjected to GC-MS analysis revealed a range of antifungal bioactive compounds with hexanedioic acid, dioctyl ester (16.57%), and oleic acid trimethylsilyl ester (12.41%) being predominant compounds. In in vitro studies, it was noticed that the T. harzianum strain was compatible with mustard cake, and hence used as a growth substrate for its mass multiplication. SEM analysis revealed no distortion in spores and mycelium of T. harzianum grown on the mustard cake. Further, seed germination assay suggested the optimum concentration of mustard cake (10%) supporting the germination rate and economics of formulation development. In in planta assay, the combination of biocontrol agent and mustard cake showed 48% disease reduction, and ~ 40% with T. harzianum alone in comparison to untreated control. Also, the combination of mustard cake and T. harzianum significantly enhanced the growth parameters of S. lycopersicum. The findings of the current study identified an environmentally friendly alternative for mitigation of Fusarium wilt, thereby providing a sustainable option for mitigation of wilt disease and enhancement of plant health.

Efficacies of bacterial and fungal isolates in biocontrol of Botrytis cinerea and Pseudomonas syringae pv. tomato and growth promotion in tomato do not correlate

There is a need to develop more biological control agents to fulfil the increasing demand for biological crop protection. Testing for consistent efficacy in disease control under the relevant range of environmental conditions is one of the most demanding steps during screening programs. Bioassays were conducted to target three major diseases of tomato, stem canker caused by Botrytis cinerea, leaf spot caused by B. cinerea, and bacterial spot caused by Pseudomonas syringae pv. tomato, and to assess possible growth promotion of tomato seedlings. Nine quantitative screening approaches were analyzed for a test panel of approximately 100 isolates of bacteria and fungi, all obtained from tomato, and several known antagonists as reference isolates. Even with such a limited number of isolates promising antagonists, partly not yet described as antagonists, could be selected for control of the targeted diseases when labor and resource demanding in planta bioassays had been applied. Also some promising isolates enhancing seedling development could be identified. Independent screening assays for the different traits were needed since no correlation between the different traits were found. Attempts to simplify screening assays to high-throughput systems failed since there were no positive correlations with in planta bioassays. In conclusion, the often suggested first screening rounds using in vitro tests for huge numbers of isolates followed by in planta testing of a selected group of candidates, e.g. those with high in vitro production of certain secondary metabolites or biosurfactants, may not exploit the entire potential of antagonists. Especially antagonists combining various modes of action may be excluded by in vitro screening with a bias on a specific mode of action. Therefore, independent in planta assays are proposed to screen against different pathogens and for growth promotion.

Nematicidal Efficacy of Milbemectin against Root-Knot Nematodes.

The nematicidal efficacy of milbemectin and its commercial formulate Milbeknock® on (i) egg hatching, (ii) juvenile motility and (iii) infective capacity of root-knot nematodes was evaluated in vitro and in planta assays. Serial dilutions of pure milbemectin were tested against nematode eggs and juveniles and lethal concentrations LC50 and LC90 calculated. Exposure of egg masses to milbemectin at a concentration of 30 μg/mL for 72 h reduced egg hatching by 52%. The increase in exposure time to 240 h did not increase the egg hatching inhibition at the highest concentration 30 μg/mL (53%) but reduced egg hatching at 15 and 7 μg/mL by 35 and 24%, respectively, when compared to untreated controls. The inhibitory effect of milbemectin on juvenile motility ranged from 41 to 87% depending on its concentration, and this effect was persistent after rinsing the juveniles in water. The probabilistic dose–response model indicated that lethal concentrations of milbemectin for juvenile motility were LC50: 7.4 μg/mL and LC90: 29.9 μg/mL. The pre-plant application of Milbeknock® to soils infested with the nematode reduced its infective capacity by 98–99% compared to untreated soils in pot experiments. Milbeknock® reduced nematode soil population densities by 50–60% in natural infestations under field conditions. Milbemectin shows a high level of efficacy against root-knot nematodes as it reduces egg hatching, persistently immobilizes nematode juveniles, and reduces tomato root infection.

Endophytes from Gnetum gnemon L. can protect seedlings against the infection of phytopathogenic bacterium Ralstonia solanacearum as well as promote plant growth in tomato

Endophytes are beneficial plant microbes which help the plants by producing various plant growth promoting substances and also by acting as biocontrol agents against various plant pathogens. In the present study, evaluation of endophytic bacteria isolated from Gnetum gnemon, an ethnomedicinal plant was carried out for their plant growth promoting (PGP) activity and antagonistic potential against bacterial wilt pathogen Ralstonia solanacearum. Initially a total of 40 endophytic bacteria were isolated which were clustered into 13 groups based on RFLP and BOX-PCR fingerprinting. These 13 representative isolates belonged to different genera of Bacillus (9), Solibacillus (1), Staphylococcus (2) and Caballeronia (1). Among them, six isolates were positive for production of IAA, the value of which ranged from 11.16 to 27.63 μg mL−1. Phosphate solubilisation in the range of 106.4 to –212.7 μg mL−1 was shown by three isolates. Eight isolates producedammonia, the value of which ranged from 1.3 to 6.1 μmol mL−1. All tested isolates were positive for siderophore production. For extracellular enzyme production, 7 isolates were positive for protease, 8 for cellulase and 10 for amylase production. The isolates were also tested for their antagonistic activity against R. solanacearum in vitro and in planta assay using tomato seedlings. Staphylococcus warneri GL1 showed the highest biocontrol efficacy of 77.67 % followed by Bacillus velezensis GL3 i.e. 70.1 %. R. solanacearum antagonistic isolates were analysed for the presence of antimicrobial peptide biosynthesis genes bmyB, srfAA, fenD and ituC. All the antagonistic isolates showed the presence of all four genes, except the isolate Bacillus velezensis GMC2, where the gene for fengycin synthetase (fenD) was absent. Based on in vitro PGP traits, three isolates Bacillus velezensis GL3, Bacillus atrophaeus GMC1 and Bacillus megaterium GS2 were selected, these three endophytic bacteria individually and their consortia were tested for in planta PGP activities in tomato plants. Application of Bacillus velezensis GL3 alone and consortia of three isolates showed significant improvement in growth parameters such as shoot length, fresh weight and dry weight in a pot experiment. Colonization of endosphere of treated tomato seedlings by the endophytic isolate Bacillus velezensis GL3 was confirmed by visualization of colony morphology and BOX-PCR fingerprinting. Our study highlights the potential of endophytes associated with unexplored plants like G. gnemon for development of bioformulation aimed at enhancing plant growth and bacterial wilt disease control.

The Hsp90 Inhibitor, Monorden, Is a Promising Lead Compound for the Development of Novel Fungicides.

Endophytic fungi are great resources for the identification of useful natural products such as antimicrobial agents. In this study, we performed the antifungal screening of various plant endophytic fungi against the dollar spot pathogen Sclerotinia homoeocarpa and finally selected Humicola sp. JS-0112 as a potential biocontrol agent. The bioactive compound produced by the strain JS-0112 was identified as monorden known as an inhibitor of heat shock protein 90 (Hsp90). Monorden exhibited strong antagonistic activity against most tested plant pathogenic fungi particularly against tree pathogens and oomycetes with the minimum inhibitory concentration values less than 2.5 μg mL–1. Extensive in planta assays revealed that monorden effectively suppressed the development of several important plant diseases such as rice blast, rice sheath blight, wheat leaf rust, creeping bentgrass dollar spot, and cucumber damping-off. Especially, it showed much stronger disease control efficacy against cucumber damping-off than a synthetic fungicide chlorothalonil. Subsequent molecular genetic analysis of fission yeast and Fusarium graminearum suggested that Hsp90 is a major inhibitory target of monorden, and sequence variation among fungal Hsp90 is a determinant for the dissimilar monorden sensitivity of fungi. This is the first report dealing with the disease control efficacy and antifungal mechanism of monorden against fungal plant diseases and we believe that monorden can be used as a lead molecule for developing novel fungicides with new action mechanism for the control of plant diseases caused by fungi and oomycetes.

Mode of Action of the Catalytic Site in the N-Terminal Ribosome-Inactivating Domain of JIP60.

Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley (Hordeum vulgare) and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in Nicotiana benthamiana that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163-185) is removed, initiates cell death, leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in N. benthamiana, as well as through an in vitro translation-inactivation assay. We found that Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.

In vitro and in planta efficacy studies on T6 generation of transgenic Rojolele rice lines against the rice yellow stem borer (Scirpophaga incertulas Walker)

One of the most destructive pests in cultivated rice (Oryza sativa L.) is the rice yellow stem borer (YSB; Scirpophaga incertulas Walker). YSB attacks rice plant throughout its life span, with the most devastating being during mature stage that causes a symptom called whiteheads. Every 1% of whiteheads symptoms resulted in 1% rice production losses. Planting YSB-resistant cultivars is a good strategy besides environmentally friendly. However, since the unavailability of YSB-resistant gene in rice germplasm, genetic engineering of rice to express Cry toxin from Bacillus thuringiensis (Bt) that is toxic to YSB is an alternative. Agrobacterium-mediated transformation has been conducted to introduce cry1B gene from Bt into a local javanica rice cultivar, Rojolele. The recombinant plasmid contains cry1B gene driven by wound-inducible promoter from maize proteinase inhibitor (mpi) gene and hygromycin phosphotransferase (hpt) selectable marker gene for transgenic plants selection. The presence of cry1B gene in transgenic rice plants was identified by PCR analysis. Four homozygous transgenic rice lines harboring cry1B gene have been obtained. The purpose of this experiment was to determine the efficacy of cry1B gene in homozygous transgenic rice lines against YSB at vegetative stage, in vitro and in planta at T6 generation. In vitro assay was performed in aerated Petri dishes using leaf samples under growth room condition, meanwhile in planta assay was done by subjecting the four homozygous transgenic rice lines to YSB under greenhouse condition. The results showed that the four homozygous transgenic rice lines tested were highly resistant to YSB compared to the wild-type Rojolele rice.

Role of the XylA gene, encoding a cell wall degrading enzyme, during common wheat, durum wheat and barley colonization by Fusarium graminearum

Fusarium graminearum is the main causal agent of fusarium head blight (FHB) of wheat and barley. This filamentous fungus is able to produce hydrolytic enzymes, such as xylanases, that cause cell wall degradation, permitting host colonization. This study investigated the role of the F. graminearum XylA (FGSG_10999) gene during infection, using a knockout mutant in strain CS3005. Assays were carried out on common wheat, durum wheat and barley to compare virulence of a XylA knockout to that of wild type strain. These assays were conducted on wheat and barley seedling roots, seedling stem bases and heads. Furthermore, additional in vitro experiments were conducted to investigate the role of XylA gene in the utilisation of D-xylose, the main component of cereals cell wall. In planta assays showed the importance of XylA gene for F. graminearum virulence towards its main hosts. A positive correlation between symptom incidence and fungal biomass development was also observed for both the wild type and the knockout strains. Finally, gene expression studies performed in a liquid medium enriched with D-xylose, a known xylanase inducer in other fungi, showed that the absence of the gene in the FGSG_10999 locus was not compensated by two other F. graminearum xylanase encoding genes analysed (loci FGSG_06445 and FGSG_11478).

Pseudomonas chlororaphis CP07 strain reduces disease severity caused by Phytophthora palmivora in genotypes of Theobroma cacao

Black pod rot is the most important disease affecting Theobroma cacao in Cuba. Plant growth promoting bacteria (PGPB) are considered an alternative for controlling plant disease in the context of the integrated management. The present work was aimed to evaluate Pseudomonas chlororaphis strain CP07, isolated from T. cacao rhizosphere, as potential PGPB against P. palmivora in cacao plants cultivated under controlled conditions. Three genotypes of Cuban traditional cacao of the group Trinitario and the genotype UF 677 were used. The effect of CP07 was evaluated by using detached leaf tests and in planta assays with plants obtained in vitro from both seeds and micrografting. Inoculation of P. palmivora in control plants showed that genotypes EICB-371, EICB-384 and UF 677 were highly susceptible to the disease (71.4 to 99% disease severity) while EICB-385 was significantly less susceptible (39 to 54.5%). In EICB-371 and EICB-385 genotypes, disease symptoms were significantly reduced in presence of CP07 compared to the control plants in all assays. In contrast, for EICB-384 and UF 677 genotypes, there was not reduction of disease severity in plants pre-treated with CP07. Results show the protective effect of treating cacao plants with CP07 against P. palmivora. Plant genotype significantly influenced the protective ability of bacteria affecting the CP07-mediated disease control. Results suggest that depending on cacao genotype, CP07 has a potential for inducing plant defense against P. palmivora which can be used in the control of black pod rot.

The growth rate of apple bitter rot lesion, caused by Colletotrichum spp., is affected by temperature, fungal species, and cultivar.

The growth and symptom development of two fungal species, Colletotrichum siamense, and C. fioriniae were examined using in vitro and in planta assays. In a plate assay, C. siamense grew fastest at 30 °C and at 25 °C, but C. fioriniae grew slower at 30 °C than at 25 °C. With apple inoculation assay, the mean lesion diameters at 30 °C were significantly larger (P ≤ 0.05) than that at 20 °C on a cultivar Ida Red for both fungal species. On the other hand, the mean lesion diameter on a cultivar Golden Delicious was significantly larger at 20 °C than at 30 °C for both fungal species. Therefore, the rate of lesion development was influenced not only by fungal species and incubation temperature but also by apple cultivar. In this study, cultivar ‘Ida red’ was found to be more susceptible to C. siamense under warm environmental condition. Although both species were able to grow at 10 °C in the plate assay, no disease symptoms were developed at 10 °C with the inoculation assay. Although the numbers of cultivar and fungal species are small, to our knowledge, this is the first report to describe potential interactive effect among Colletotrichum species, temperature, and apple cultivar on the rate of bitter rot symptom development.

Target and non-target effects of dual inoculation of biocontrol agents against Fusarium wilt in Solanum lycopersicum

Bacillus subtilis and Trichoderma harzianum are known for their antagonistic potential against several fungal phytopathogens. However, limited studies have assessed the non-target effects of these biocontrol agents. The present study aimed to investigate the antagonistic efficacy of B. subtilis (MTCC 2274) and T. harzianum (MTCC 3928) in mitigation of Fusarium wilt, and to study their non-target effects on the rhizospheric bacterial community in tomato. In planta assay revealed that the consortium of B. subtilis and T. harzianum exhibited the highest disease reduction (56%), whereas with chemical control it was 48.7% as compared to untreated control. Also, the impact of the dual inoculation on the bacterial community was qualitatively and quantitatively monitored by PCR-denaturing gradient gel electrophoresis (DGGE) and qPCR, respectively by employing 16S rRNA as a marker. DGGE profiles generated from rhizospheric samples from treatment (90 DAS) with T. harzianum and B. subtilis, and chemical treatment formed a cluster with 72% similarity. The phylogenetic analysis of excised bands showed the presence of ecologically important species (Desulfotomaculum thermoacetoxidans, Microcoleus sp., Thermoactinomyces sp. and Nocardioides sp.) in soil treated with dual inoculation. Furthermore, qPCR of rhizospheric soil (90 DAS) treated with both inoculants showed a 16S rRNA gene copy number/g dry soil of 2.9 × 104 in comparison to untreated control (8.8 × 103) that confirmed the positive change in bacterial abundance. These results demonstrated the integrated role of biocontrol agents towards soil health improvement along with mitigation of Fusarium wilt.

Characterization of Burkholderia glumae Putative Virulence Factor 11 (PVF11) via Yeast Two-Hybrid Interaction and Phenotypic Analysis.

In this study, PVF11 was selected among 20 candidate pathogenesis-related genes in Burkholderia glumae based on its effect on virulence to rice. PVF11 was found to interact with several plant defense-related WRKY proteins as evidenced through yeast-two hybrid analysis (Y2H). Moreover, PVF11 showed interactions with abiotic and biotic stress response-related rice proteins, as shown by genome-wide Y2H screening employing PVF11 and a cDNA library from B. glumae-infected rice. To confirm the effect of PVF11 on B. glumae virulence, in planta assays were conducted at different stages of rice growth. As a result, a PVF11-defective mutant showed reduced virulence in rice seedlings and stems but not in rice panicles, indicating that PVF11 involvement in B. glumae virulence in rice is stage-dependent.

GhNAC83 inhibits corm dormancy release by regulating ABA signaling and cytokinin biosynthesis in Gladiolus hybridus.

Corm dormancy is an important trait for breeding in many bulb flowers, including the most cultivated Gladiolus hybridus. Gladiolus corms are modified underground stems that function as storage organs and remain dormant to survive adverse environmental conditions. Unlike seed dormancy, not much is known about corm dormancy. Here, we characterize the mechanism of corm dormancy release (CDR) in Gladiolus. We identified an important ABA (abscisic acid) signaling regulator, GhPP2C1 (protein phosphatase 2C1), by transcriptome analysis of CDR. GhPP2C1 expression increased during CDR, and silencing of GhPP2C1 expression in dormant cormels delayed CDR. Furthermore, we show that GhPP2C1 expression is directly regulated by GhNAC83, which was identified by yeast one-hybrid library screening. In planta assays show that GhNAC83 is a negative regulator of GhPP2C1, and silencing of GhNAC83 promoted CDR. As expected, silencing of GhNAC83 decreased the ABA level, but also dramatically increased cytokinin (CK; zeatin) content in cormels. Binding assays demonstrate that GhNAC83 associates with the GhIPT (ISOPENTENYLTRANSFERASE) promoter and negatively regulates zeatin biosynthesis. Taken together, our results reveal that GhNAC83 promotes ABA signaling and synthesis, and inhibits CK biosynthesis pathways, thereby inhibiting CDR. These findings demonstrate that GhNAC83 regulates the ABA and CK pathways, and therefore controls corm dormancy.

Biological control of bacterial plant diseases with Lactobacillus plantarum strains selected for their broad-spectrum activity.

The use of lactic acid bacteria (LAB) to control multiple pathogens that affect different crops was studied, namely, Pseudomonas syringae pv. actinidiae in kiwifruit, Xanthomonas arboricola pv. pruni in Prunus and Xanthomonas fragariae in strawberry. A screening procedure based on in vitro and in planta assays of the three bacterial pathogens was successful in selecting potential LAB strains as biological control agents. The antagonistic activity of 55 strains was first tested in vitro and the strains Lactobacillus plantarum CC100, PM411 and TC92, and Leuconostoc mesenteroides CM160 and CM209 were selected because of their broad‐spectrum activity. The biocontrol efficacy of the selected strains was assessed using a multiple‐pathosystem approach in greenhouse conditions. L. plantarum PM411 and TC92 prevented all three pathogens from infecting their corresponding plant hosts. In addition, the biocontrol performance of PM411 and TC92 was comparable to the reference products (Bacillus amyloliquefaciens D747, Bacillus subtilis QST713, chitosan, acibenzolar‐S‐methyl, copper and kasugamycin) in semi‐field and field experiments. The in vitro inhibitory mechanism of PM411 and TC92 is based, at least in part, on a pH lowering effect and the production of lactic acid. Moreover, both strains showed similar survival rates on leaf surfaces. PM411 and TC92 can easily be distinguished because of their different multilocus sequence typing and random amplified polymorphic DNA profiles.