Challenge Inoculation Research Articles

Role of non-conventional chemicals in biochemical defence against charcoal rot of sesame incited by Macrophomina phaseolina (Tassi) Goid

Charcoal rot of sesame (Sesamum indicum) incited by Macrophomina phaseolina (Tassi) Goid. is an economically important disease in India and worldwide. Seed and soil borne nature, non-specific, wide host range of pathogen, difficult to manage with fungicides and absence of complete genetic resistance impelled to biochemical defence analysis against this pathogen. Treatment with non-conventional chemicals induces defence against pathogen in the form biochemical changes in plant. Hence, the present investigation was carried out to know the impact of non-conventional chemicals on changes in defence biochemical parameters in plant. As the concentration of different non conventional chemicals increased from 50, 100 and 200 ppm there was reduction in incidence of charcoal rot disease. Maximum induction of resistance was recorded by the application of salicylic acid at 200 ppm concentration after challenge inoculation with M. phaseolina followed by indole acetic acid, indole butyric acid, acetyl salicylic acid, riboflavin and thiamine. The activity of total phenols and sugar content was reached at peak after six days of challenge inoculation with the pathogen and subsequently activity of these parameters slightly declined after six days of inoculation in all tested non-conventional chemicals. At each concentration, SA showed maximum induction of total phenol and total sugar followed by IAA, IBA, ASA, thiamine and riboflavin. However, sesame variety HT 2 showed higher induction of biochemical activities and lower disease incidence of charcoal rot as compared to HT 1.

Acibenzolar-S-methyl and β-amino butyric acid-induced upregulation of biochemical defense against Colletotrichum capsici infection in chilli (Capsicum annuum)

Abiotic inducers (AI) are synthetic/natural compound, induces systemic resistance in plants against pathogens. Present study aimed to evaluate the foliar application of AI [Acibenzolar-S-Methyl (ASM), and β-Amino Butyric Acid (BABA)] in managing chilli anthracnose under greenhouse conditions. Foliar applications of ASM-5 μg/ml and BABA-10 mM significantly decreases disease incidence to 13.15% and 14.1% respectively, when compared to respective controls (65% and 65.34%). AI treated seedlings recorded maximum H2O2 accumulation at 9 h (ASM) and 12 h (BABA) after challenged with pathogen. In AI treated seedlings, activity of defense-related enzymes (Phenylalanine ammonia-lyase, Peroxidase, Polyphenol oxidase, Lipoxygenase, and chitinase) was upregulated after challenge inoculation. Similarly, maximum phenolic (μg gallic acid/g tissue) accumulation of 147 and 71.2 was recorded with ASM and BABA treatment, respectively. The AI induced changes in biochemical parameters are in correlation with reduced disease incidence. Hence, AI can use as alternative, safe and eco-friendly candidate to manage chilli anthracnose.

Bio-priming of rice seeds with novel bacterial strains, for management of seedborne Magnaporthe oryzae L.

Rice blast disease caused by Magnaporthe oryzae is a seed borne which occurs in all rice growing regions of the country (India) causing yield losses up to 40%. In the present study, we report the efficacy of different bacterial strains through seed priming in reducing the blast disease severity under in vitro, in vivo and field conditions. In vitro evaluation revealed that the Bacillus amyloliquefaciens strain UASBR9 and Serratia marcescens strain UASBR4 were found most effective in reducing disease severity (84.28%) compared to reference cultures Bacillus subtilis RBs-1 (72.00%) and Pseudomonas fluorescens RPf-1 (55.24%). The least disease severity and highest seed germination was recorded in seeds bio-primed with B. amyloliquefaciens UASBR9 (0.96 and 98.00%) under in vivo. Bio-priming also activated the induced systemic resistance as evident in the increased activities of peroxidase (0.563/min/g) and polyphenol oxidase (0.825/min/g) defense-related enzymes in comparison with untreated control in rice seedlings upon challenge inoculation with M. oryzae. Field studies showed that seeds bio-primed with B. amyloliquefaciens UASBR9 recorded lowest disease severity (7.0%) of rice blast and increased the grain yield (0.67 kg/m2). Our results indicated that B. amyloliquefaciens UASBR9 could be served as plant growth promoter and an environmental friendly alternative for chemical pesticides to manage rice blast disease.

Role of lipopolysaccaride extracted from Alcaligenes faecalis as elicitor for the induction of plant defense against fusarium wilt

In the present investigation, emphasis was placed on the efficacy of lipopolysaccharide (LPS) extracted from the plant beneficial rhizobacterium Alcaligenes faecalis MSS8 for the induction of disease resistance by enhancing immune response in plants against phytopathogen Fusarium oxysporum f. sp. lycopersici (FOL). Characterization and proficiency of the rhizobacterial LPS for the induced defense response in plant against FOL was revealed by the gel activity staining of Peroxidase (PO) and Polyphenol oxidase (PPO) isoforms as compared to the untreated and only FOL treated plants. It has been observed that after 48 h of FOL challenge inoculation, in the LPS pretreated plant there was significant induction of PO and PPO isoforms. Furthermore, RT-PCR analysis for the study of relative gene expression of pathogenesis related (PR) proteins confirms the result of gel activity staining. It has been observed that expression level of LeChi9, LeGluB, LePR1a was significantly upregulated in LPS treated plants 24 h after FOL inoculation and maximum level was observed at 48 h as compared to the control and FOL treated plants. This clearly indicates the role of LPS extracted from A. faecalis as a potential biocontrol agent against fusarium wilt.

Induction of defense-related genes in tomato plants after treatments with the biocontrol agents Pseudomonas chlororaphis ToZa7 and Clonostachys rosea IK726.

Pseudomonas chlororaphis ToZa7 is a promising biocontrol agent possessing valuable characteristics and reducing disease severity caused by Fusarium oxysporum f. sp. radicis-lycopersici (Forl) in tomato. In this study, the strain's ability to induce three pathogenesis-related (PR) genes (PR-1a, GLUA, and CHI3) in tomato, was studied using quantitative reverse transcription PCR. The genes PR-1a and GLUA were up-regulated after 120h exposure to P. chororaphis ToZa7 (15.22- and 13.11-fold, respectively), as compared to the untreated control, without challenge inoculation by the pathogen. To study the effects of individual or combined application of P. chororaphis ToZa7 and the compatible biocontrol fungus Clonostachys rosea IK726, challenged with the pathogen, the expression patterns of the above three PR genes were monitored, in tomato roots. Expression of PR1-a was noteworthy, especially 48h after challenge inoculation, when C. rosea IK726 alone or in combination with P. chororaphis, ToZa7 was pre-inoculated on tomato roots (38.53-fold and 53.74-fold, respectively). Expression of PR1-a, 72h after challenge inoculation, was the highest in P. chororaphis ToZa7, among biocontrol treatments. Expression of CHI3 was much lower, while up-regulation of GLUA was overall not observed. Confocal laser scanning microscopy of intact tomato roots and bacterial counts of superficially disinfected roots revealed, for the first time, that P. chororaphis ToZa7 colonizes the exterior as well as the internal tissues.

Identification of Resistance to Citrus Black Spot Using a Novel In-field Inoculation Assay

Citrus black spot is an important fungal disease of citrus resulting in fruit drop and rind blemish in tropical and subtropical production areas. The disease is incited by the fungus Phyllosticta citricarpa (McAlpine) van der Aa (synonym: Guignardia citricarpa Kiely), with control currently relying on the application of fungicides. Because the presence and expression of resistance is poorly understood, we sought to develop a method for inoculating fruit in the field that gives reproducible symptoms of citrus black spot consistent with natural field infection. We subsequently validated this method by screening 49 citrus accessions and characterized their qualitative expression of citrus black spot symptoms. Challenge inoculations were undertaken with a known isolate of P. citricarpa, and control fruit were inoculated with water or the endophyte P. paracapitalensis Guarnaccia & Crous. Our results showed that all mandarin, sweet orange, lemon and papeda types were susceptible; pummelo, lime, and sour orange types expressed immunity; while various hybrids were susceptible, resistant and immune. Hybrid progeny from crosses using pummelo [Citrus maxima (Burm.) Merr.] as a parent showed preliminary evidence of segregation for citrus black spot immunity. The implications of these results to achieve genetic improvement for citrus black spot resistance in citrus breeding programs are discussed.

Effects of challenge dose and inoculation route of the virulent Neospora caninum Nc-Spain7 isolate in pregnant cattle at mid-gestation

Parameters such as pathogen dose and inoculation route are paramount in animal models when studying disease pathogenesis. Here, clinical findings, including foetal mortality, parasite transmission rates and lesion severity, and immune responses were evaluated in Asturiana pregnant heifers at day 110 of gestation challenged with a virulent (Nc-Spain7) Neospora caninum isolate. Four different doses of parasite tachyzoites were inoculated intravenously (IV1, 107 parasites, n = 6; IV2, 105, n = 6; IV3, 103, n = 6; and IV4, 102, n = 5), and the subcutaneous (SC) inoculation route was also assessed for the dose of 105 tachyzoites (SC, n = 6). In addition, a control group (n = 4 pregnant heifers) was evaluated. Foetal death was observed in all infected groups from 25 to 62 days post-infection, varying with the dose (IV1:4/6, IV2:3/6; IV4:2/5, IV3:1/6), and was three times less frequently associated with the SC route than IV inoculation (1/6 vs. 3/6). A dose-dependent effect for parasite loads in placental and foetal brain tissues was also detected. After SC challenge, a reduced number of tachyzoites were able to reach foetal brain tissues, and no lesions were observed. In calves, specific IgG responses in precolostral sera were mainly associated with high-dose groups (IV1 [100.0%] and IV2 [66.7%]), and cerebral parasite DNA detection was scarce (3/18). In dams, IFN-γ production and the dynamics of anti-N. caninum IgG antibodies varied with the dose, and the cell-mediated immune response was also found to be route-dependent. Our results confirm the influence of parasite dose and inoculation route on the outcome and dynamics of bovine neosporosis at mid-gestation.

Salmonella recovery from chicken bone marrow and cecal counts differ by pathogen challenge method.

Mechanically separated chicken (MSC) may be incorporated into other further processed foods, and has been identified as a transmission vehicle in human foodborne disease outbreaks involving the pathogen Salmonella enterica. Chickens possess pneumatic bones producing marrow, which may become infected with Salmonella and present a mechanism for Salmonella contamination of MSC. The primary objective of the current study was to ascertain whether chicken bone marrow was susceptible to Salmonella infection as a function of pathogen challenge method. Additionally, this study sought to determine the impact of Salmonella challenge site and inoculation on subsequent numbers of the microorganism in the cecum at 3, 6 and 9 d post-challenge. In this study, 30-day-old birds (Gallus domesticus) were separately challenged with S. enterica by one of the 6 experimental treatment methods: oral or endo-tracheal gavage, and transdermal scratch challenge on the breast or back muscles, with or without feathers (N = 150). Differing Salmonella recovery rates were detected in bone marrow from euthanized birds (P = 0.0015); oral and endo-tracheal gavage produced Salmonella recovery frequencies of 10% and 20%, respectively. Counts of Salmonella from chicken cecal samples statistically differed as a function of challenge method (P = 0.032); the highest numbers of Salmonella in cecal samples were obtained from orally gavaged birds (3.0 log10 cfu/g). Study results demonstrate the potential for chickens to contract short-term systemic infection following Salmonella challenge using methods of pathogen challenge simulating aerosol inhalation, ingestion, or wounding. Salmonella entry into chicken bone marrow may result in decreased likelihood of Salmonella performance standard compliance and increased pathogen transmission risk to consumers.

Efficacy of plant growth promoting bacteria Bacillus amyloliquefaciens B9601-Y2 for biocontrol of southern corn leaf blight

Southern corn leaf blight caused by Bipolaris maydis is one of the major threat to maize production worldwide. In this study, Bacillus amyloliquefaciens B9601-Y2, previously isolated from wheat rhizosphere, exhibited antagonistic effect against several important phytopathogens, solubilized mineral phosphate, potassium and was able to grow without nitrogen under in in vitro conditions. To further evaluate its potential agricultural use, maize pot and field experiments were performed to evaluate the plant growth-promotion and southern leaf blight suppression of B9601-Y2 culture. The results showed that B9601-Y2 significantly promoted maize seedling growth, improved several enzymes associated with soil quality, as well as increased chlorophyll content. After challenge inoculation 10 days with Bipolaris maydis, the causal agent of southern leaf blight, B9601-Y2-treated maize plants showed 61.38% decrease in disease index. To determine the antagonistic mechanism of B9601-Y2, its colonization pattern in the rhizosphere and maize tissue were also investigated using a GFP marker. The results indicated that B9601-Y2 predominantly colonized the elongation zones of primary roots, root hairs and the junctions between roots, subsequently entering the stems and leaves. The population dynamics of GFP-tagged B9601-Y2 in the rhizosphere and on maize roots were monitored. B9601-Y2 significantly reduced southern leaf blight severity in maize and increased its marketable yield under field conditions. The present study demonstrated that B9601-Y2 may be developed as a promising biocontrol and plant-growth-promoting agent due to its enhanced nutrient solubilization and disease suppression traits.

Seed priming induced blast disease resistance in finger millet plants through phenylpropanoid metabolic pathway

Phenylpropanoids contribute to all aspects of plant responses to biotic and abiotic stimuli. The lignin biosynthesis by phenylpropanoid pathway induces cell wall strengthening, a barrier against fungal penetration and progression. In the present study, two fluorescent Pseudomonas isolates JUPC113 and JUPW121 from cotton and wheat rhizospheres respectively were used for seed priming in greenhouse studies along with the unprimed seeds as control. Plants were assessed for disease symptoms from 10 to 20d of challenge inoculation with the blast pathogen Magnaporthe grisea (MTCC-1477). Pronounced increase in disease resistance was seen in primed plants; JUPW121 (0.21 ± 0.04) and JUPC113 (0.27 ± 0.02) compared with the mock control plants with maximum disease incidence rate of 2.67 ± 0.33. Spectrophotometric estimation of the different enzymes involved in lignin biosynthesis, viz., phenylalanine ammonia lyase (PAL; EC 4.3.1.24) and tyrosine ammonia lyase (TAL; EC 4.3.1.25) showed an increased activity in JUPC113-primed plants at the initial 6 and 12 hpi in comparison with the control. The biochemical studies were validated by the histochemical (lignin) and metabolomic (phenolic compounds and amino acids) studies. The disease incidence rate was also low when compared to the primed and mock control plants which was validated by biochemical estimation of the enzyme activities. The present study presented the experimental evidence on induction of disease resistance in finger millet against the pathogen by accumulation of host defense compounds during early hours post challenge. Hence these isolates can be used in further studies of seed priming for disease resistance against different plant pathogens.

Spatial characteristics of volatile communication in lodgepole pine trees: Evidence of kin recognition and intra-species support.

Plant interactions using volatile organic compounds, particularly in the context of kin recognition have received considerable attention in recent years, but several discrepancies and conflicting results have restricted our understanding. We propose that some of these discrepancies in literature are in part due to integral spatial characteristics of sites, and plant attributes. Chemotypic plasticity is commonly used to characterize kin, particularly in conifers. We studied constitutive and induced monoterpene chemotypes of non-attacked lodgepole pine trees within 30 m radii of pine trees attacked by mountain pine beetle. We tested the effects of volatile compounds emitted from the attacked trees on the non-attacked trees by challenge inoculations with a mountain pine beetle associated fungus. We found no relationship between constitutive monoterpene concentrations of the non-attacked trees and distance or direction from the attacked trees or site aspects. In contrast, the effects of volatile compounds were evident after inoculations, depending on distance from the attacked trees and site aspects. However, these interactions only emerged among chemotypically related trees. These results suggest that plants discriminate between chemical cues from kin and strangers, and the emitters likely aid only chemotypically related plants by emitting specific blends of volatiles that can only be deciphered by the receiving kin. These results further demonstrate the importance of incorporating spatial characteristics of sites and plant attributes in studies aimed at investigating intra-species interactions using volatile organic compounds.

Piriformospora indica-primed transcriptional reprogramming induces defense response against early blight in tomato

Piriformospora indica is an adaptable mycorrhiza-like fungus belonging to the Sebacinales order that can colonize roots of a wide range of plant species. Studies have shown that P. indica improves growth and enhances systemic defense against pathogens in host plants. However, the mechanism(s) through which these effects occur remain unclear. Therefore to gain more insight into the molecular basis of P. indica induced resistance, cDNA-AFLP (Amplified fragment length polymorphism) based transcript profiling was done to identify differentially expressed genes in P. indica-colonized tomato plants infected with Alternaria solani. Our results demonstrated that pre-colonization of tomato roots with P. indica systemically induced resistance against early blight. Transcript profiling of P. indica pre-colonized tomato plants revealed systemic modulation of several key components of signaling network transcriptional regulators including CBL-interacting protein kinase (CIPK), Mitogen activated protein kinases (MPKs), Lipid transfer proteins (LTPs), WRKY1, ethylene responsive transcription factors (ERF), and Jasmonate Zim Domain 1 (JAZ1), a negative regulator of jasmonic acid (JA) signaling. Expression of downstream defense related genes like Thaumatin-like protein, β-1, 3-glucanase and chitinases was also affected in leaves upon challenge inoculation with pathogen. Interestingly, P. indica pre-colonization alone was unable to induce transcript levels for most of the genes studied. However, pathogen attack on P. indica pre-colonized plants induced strong defense responses. In conclusion, P. indica induces transcriptome reprogramming in a manner that allows rapid and efficient activation of JA/ET (jasmonic acid/ethylene)-mediated basal defenses against pathogen infection by altering the expression of JA/ET related genes. P. indica colonization appears to potentiate the complete signal transduction cascade leading to the systemic expression of defense genes against foliar pathogens. It thus presents itself as a potential and sustainable method of activating multiple components of defense signaling thereby conveying durable horizontal defense against a range of pathogens.

Mixed PGPR consortium: an effective modulator of antioxidant network for management of collar rot in cauliflower

Sclerotinia sclerotiorum is a cosmopolitan fungal pathogen causing stem and collar watery soft rot of cauliflower. Previous management of this disease with chemical pesticides caused hazardous results which lead to use of more eco-friendly microbial approaches. In the present study, consortia of Trichoderma harzianum TNHU27 and Pseudomonas aeruginosa PJHU15 were assessed for their ability in controlling Sclerotinia rot in cauliflower. The seedlings of cauliflower were challenged singly and in consortium with these two compatible microbes. The plants were evaluated upon challenge inoculation with S. sclerotiorum for changes in total phenolic content, the activity of defense and antioxidative enzymes. The microbial consortium comprising of T. harzianum and P. aeruginosa significantly enhanced the defense responses of the plant in comparison to pathogen challenged and unchallenged control. The study elucidates that plant beneficial microbes in the consortium may provide superior protection by induction of faster and enhanced defense responses in comparison to unchallenged and single microbe challenged plants under pathogen challenged conditions.

Induction of systemic resistance in turmeric by rhizospheric isolate Trichoderma asperellum against rhizome rot disease

The rhizospheric fungal isolates were screened for growth promotion and induced systemic resistance against rhizome rot disease in turmeric caused by Pythium aphanidermatum (Edson) Fitzp. Thirty isolates from the turmeric rhizosphere were identified by morphological characteristics and using internal transcribed spacer sequence homology. In in vitro antagonistic activity, out of 30 isolates tested only five isolates showed >70% suppression of P. aphanidermatum. The rhizospheric isolates viz., T. viride PGPFDOB-V6, Chaetomium sp. PGPFDOB-V13, T. asperellum PGPFDOB-V11, T. harzianum PGPFDOB-V22 and T. asperellum PGPFDOB-V36 also showed multiple plant growth promoting traits under in vitro studies. In greenhouse studies, rhizome treatment followed by soil application of PGPFDOB-V36 significantly increased the plant height to 87.60 cm and fresh rhizome yield/plant to 430 g. This isolate reduced the percent disease incidence (PDI) of rhizome rot to 14.2% when compared to the control 78.70%. The rhizome colonization of PGPFDOB-V36 was observed by using the confocal microscope. The selected PGPF isolates were tested for their ability to induce production of defense-related enzymes in plants. Temporal expression pattern of defense-related enzymes such as peroxidase (PO), phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and PR-protein β-1,3 glucanase in turmeric plants pretreated with PGPF isolates followed by challenge inoculation with the pathogen was studied. The defense enzymes were increased by two to three folds compared to uninoculated control. The accumulation of phenolics was higher in plants pre-treated with the PGPF isolates. The study revealed that the rhizosphere isolate PGPFDOB-V36 has promising plant growth promoting and rhizome rot suppression ability in turmeric.

Soil solarization and amelioration with calcium chloride or Bacillus licheniformis - an effective integrated strategy for the management of bacterial wilt of ginger incited by Ralstonia pseudosolanacearum

Bacterial wilt (BW) incited by Ralstonia pseudosolanacearum (Rps), is one among the most economically important and devastating disease prevalent in all the ginger growing countries. Several strategies encompassing cultural, physical and chemical means have been reported to manage bacterial wilt but with limited success. In the present study, a technology integrating physical (soil solarization), chemical (soil amelioration with calcium chloride) and biological (ginger apoplastic bacterium - Bacillus licheniformis) methods has been developed to manage BW efficiently, economically and eco-friendly. The results indicated that, CaCl2 (2 to 4%) is inhibitory to R. pseudosolanacearum under in vitro conditions. In planta evaluation under challenge inoculation showed 71%, 98% and 100% reduction in BW with B. licheniformis, 3% and 4% CaCl2, respectively. Subsequent field evaluation involving soil solarization followed by soil amelioration with CaCl2 or with B. licheniformis resulted in significant reduction in the population of R. pseudosolanacearum from 108 to 103. Further field evaluation in farmer’s plot in BW endemic regions also resulted in 100% disease suppression adopting the technology. The results emanated from the present study indicated that the technology developed which includes soil solarization along with soil amelioration with either CaCl2 3% or B. licheniformis would serve as a viable and effective integrated strategy for the management of BW in ginger.

Induced synthesis of defense molecules in tomato (Solamum lycopercicum L.) against fusarium wilt through plant extracts

Pre-application of plant extracts as resistance inducers provided protection of tomato plants and reduced wilt incidence from 84.46 - 8.40% with the minimum in garlic extract treated seedlings followed by neem and zinger extract representing 10.70 and 11.90%, respectively. Treatment with plant extracts as inducers prior to challenge inoculation sensitized the seedlings to produce increased levels of soluble protein with the maximum in garlic extract treated seedlings representing 29.5, 32.40, and 31.6 mg/g of fresh leaves of 5, 10 and 15 days of pathogen inoculation whereas, in case of control-1 and control-2 the values were 21.80, 22.50 and 22.00 mg/g and 18.98, 20.90 and 19.90 mg/g of fresh leaves, respectively. A high content of phenol, which is an indicator of first stage of defence mechanism, was also recorded maximum in garlic extract treatment as 2.09, 2.26 and 2.18 mg/g of fresh leaves against 1.50, 1.59 and 1.58 mg/g and 1.40, 1.50 and 1.46 mg/g in case of control-1 and control-2 at 5, 10 and 15 days of pathogen inoculation. Correlation coefficient between disease incidence with soluble protein (r = –0.572) and total phenol (r = –0.533) showed a negative correlation with disease incidence.

Cross protection against the watermelon strain of Papaya ringspot virus through modification of viral RNA silencing suppressor

Papaya ringspot virus watermelon strain (PRSV-W) causes huge economic losses to cucurbits production. Here, we constructed an infectious clone of PRSV-W, pCamPRSV-W, which can induce similar symptoms and accumulate to same levels as wild type virus in plants of Cucurbita pepo, Cucumis melo, Citrullus lanatus and Cucumis sativus. The green fluorescence protein gene gfp was cloned into pCamPRSV-W to produce pCamPRSV-W-GFP, which produced strong green fluorescence in systemic leaves of inoculated Cucurbita pepo, Cucumis melo, Citrullus lanatus and Cucumis sativus plants, indicating that pCamPRSV-W can be used to express foreign genes. Ten mutants of PRSV-W, obtained by site-directed mutagenesis in the RNA silencing suppressor helper-component proteinase encoding region, produced dramatically attenuated symptoms in plants of Cucumis melo. The Cucumis melo plants pre-infected with mutants K125D and G317 K showed effective protection against the challenge inoculation of wild type PRSV-W. The attenuated mutants generated in this study will be helpful for the eco-friendly control of PRSV-W.

Defensin-like peptides in wheat analyzed by whole-transcriptome sequencing: a focus on structural diversity and role in induced resistance.

Antimicrobial peptides (AMPs) are the main components of the plant innate immune system. Defensins represent the most important AMP family involved in defense and non-defense functions. In this work, global RNA sequencing and de novo transcriptome assembly were performed to explore the diversity of defensin-like (DEFL) genes in the wheat Triticum kiharae and to study their role in induced resistance (IR) mediated by the elicitor metabolites of a non-pathogenic strain FS-94 of Fusarium sambucinum. Using a combination of two pipelines for DEFL mining in transcriptome data sets, as many as 143 DEFL genes were identified in T. kiharae, the vast majority of them represent novel genes. According to the number of cysteine residues and the cysteine motif, wheat DEFLs were classified into ten groups. Classical defensins with a characteristic 8-Cys motif assigned to group 1 DEFLs represent the most abundant group comprising 52 family members. DEFLs with a characteristic 4-Cys motif CX{3,5}CX{8,17}CX{4,6}C named group 4 DEFLs previously found only in legumes were discovered in wheat. Within DEFL groups, subgroups of similar sequences originated by duplication events were isolated. Variation among DEFLs within subgroups is due to amino acid substitutions and insertions/deletions of amino acid sequences. To identify IR-related DEFL genes, transcriptional changes in DEFL gene expression during elicitor-mediated IR were monitored. Transcriptional diversity of DEFL genes in wheat seedlings in response to the fungus Fusarium oxysporum, FS-94 elicitors, and the combination of both (elicitors + fungus) was demonstrated, with specific sets of up- and down-regulated DEFL genes. DEFL expression profiling allowed us to gain insight into the mode of action of the elicitors from F. sambucinum. We discovered that the elicitors up-regulated a set of 24 DEFL genes. After challenge inoculation with F. oxysporum, another set of 22 DEFLs showed enhanced expression in IR-displaying seedlings. These DEFLs, in concert with other defense molecules, are suggested to determine enhanced resistance of elicitor-pretreated wheat seedlings. In addition to providing a better understanding of the mode of action of the elicitors from FS-94 in controlling diseases, up-regulated IR-specific DEFL genes represent novel candidates for genetic transformation of plants and development of pathogen-resistant crops.

RNA Silencing-Mediated Apple Latent Spherical Virus Vaccine in Plants.

The apple latent spherical virus (ALSV), originally isolated from an apple tree in Japan, is a small spherical virus with a diameter of 25 nm and comprises a bisegmented, single-stranded RNA genome (RNA1 and RNA2) and three different capsid proteins (Vp25, Vp20, and Vp24). The virus can experimentally infect a broad range of plants including, not only model plants (Arabidopsis thaliana and Nicotiana species) but also economically important crops such as cucumber, soybean, tomato, fruit trees, and flowers. ALSV has been used as an effective plant virus vector for virus-induced gene silencing (VIGS) to assess gene functions because the virus infects most of the host plants without showing any symptoms and induces a uniform knockout phenotype in infected plants. Moreover, the VIGS persists throughout plant growth in infected plants. Here, we show that genetically engineered ALSV vectors (ALSV vaccines) containing a partial genome sequence of pathogenic viruses display a high degree of cross-protection against the challenge inoculation of the corresponding pathogenic viruses. Treatment effects can also be expected in virus-infected plants by subsequent inoculation with ALSV vaccine.

Biopriming of micropropagated banana plants at pre- or post-BBTV inoculation stage with rhizosphere and endophytic bacteria determines their ability to induce systemic resistance against BBTV in cultivar Grand Naine

ABSTRACTRhizospheric and endophytic bacteria isolated from the roots and corms of banana were tested to find out their efficiency in controlling against banana bunchy top virus (BBTV). Bioformulations of mixtures of endophytic Bacillus pumilus and B. subtilis isolated from banana cv. Grand Naine and rhizobacterial isolate Pseudomonas fluorescens (Pf1) were found to be effective in increasing the growth and physiological parameters such as pseudostem girth and height, number of leaves, phyllochron, and leaf area in biohardened plants under greenhouse study. The consortia of bioformulation mixture of B. pumilus, B. subtilis, and P. fluorescens I showed 61.62% disease reduction over control. The defence enzymes such as peroxidase (POX), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), and total phenol were induced to an elevated level in biohardened plants. The applications of bioformulations to plants led to delay the symptom expression for 63.75 to 70.50 days compared to control after challenge inoculation with the virus in 34–67% of plants that exhibited the symptoms till 150 DAI. However, biohardening of plants with the same combinations of bacteria three days after BBTV inoculation led to express the symptoms 29.16 to 36.71 days and there was a significant decrease in plant growth parameters. Biopriming prior to BBTV infection has attributed to the enhanced plant growth and resistance against BBTV whereas, the same treatments after virus inoculation did not induce resistance. This study has proved that the time of application of consortia of bio-inoculants determines their effect of induced resistance to BBTV in micropropagated plants.