Direct Foliar Application of dsRNA Derived From the Full-Length Gene of NSs of Groundnut Bud Necrosis Virus Limits Virus Accumulation and Symptom Expression

Groundnut bud necrosis virus (GBNV) is the most significant member of the genus Orthotospovirus occurring in the Indian subcontinent. There is hardly any effective measure to prevent GBNV in crop plants. In order to develop GBNV infection prevention procedure, we examined the effect of the direct foliar application of double-stranded RNA (dsRNA) derived from the full-length NSs gene (1,320 nucleotides) of GBNV. The bacterially expressed dsRNA to the non-structural (dsNSs) gene of GBNV was purified and delivered to plants as an aqueous suspension containing 0.01% Celite for evaluating its efficacy in preventing GBNV infection in systemic host, Nicotiana benthamiana as well as in local lesion and systemic host, cowpea cv. Pusa Komal (Vigna unguiculata). The dsNSs application and challenge-inoculation were conducted in three different combinations, where plants were challenge-inoculated with GBNV a day after, immediately, and a day before the application of dsNSs. N. benthamiana plants, which were not treated with dsRNA showed severe systemic wilting and death by 9–16 days post-inoculation (dpi). The non-treated cowpea plants exhibited many chlorotic and necrotic lesions on the cotyledonary leaves followed by systemic necrosis and death of the plants by 14–16 dpi. The dsNSs treated plants in all the combinations showed significant reduction of disease severity index in both N. benthamiana and cowpea. The treatment combination where the GBNV inoculation was conducted immediately after the dsNSs treatment was found to be the most effective treatment in preventing symptom expression. The viral RNA analysis by real time PCR also showed 20 and 12.5 fold reduction of GBNV in cowpea and N. benthamiana, respectively. Our results suggest that the foliar application of dsRNA derived from the full-length NSs gene of GBNV through Celite is successful in delivering long dsRNA leading to effective prevention of GBNV infection.

Transcriptome Analysis of Sugarcane in Response to Colletotrichum Falcatum Infection Reveals Repertoire of Transcription Factors and Host Targets

Background - Sugarcane (Saccharum spp hybrid), an important C4 perennial plantation crop, globally grown for white sugar and ethanol production. Red rot caused by Colletotrichum falcatum is one of the most important threats affecting sugarcane productivity in many countries including India.Materials and Methods - Comprehensive understanding is very much needed to define their transcription level differences and their key regulatory genes during interaction of sugarcane with C. falcatum. To compute and evaluate the molecular mechanism in sugarcane, transcriptome analysis of sugarcane challenged with C. falcatum was sequenced using Hi-Seq 2500 and gene expression profiles were generated by qRT-PCR assays in both compatible and incompatible interactions after challenge inoculation of C. falcatum in sugarcane.Results - A total of 15,728,914 reads were aligned to 48,935 unigenes using BOWTIE 2; the unigenes were annotated using BLASTX and found that 39,895 unigenes were annotated and 22,025 were unigenes with respect to host species, 8,830 with respect to Colletotrichum spp and 9,040 were found to be novel genes. A total of 243 transcription factors (TFs) were found to be predicted in sugarcane challenged with C. falcatum and those TFs were divided into 45 specific families. WRKY, MYB, NAC, bHLH and AUX/IAA transcription factors were found to be abundant which are considered to be key regulators in controlling wide range of molecular events such as defense response, oxidative stimuli, host signalling and triggering disease resistance. In addition, a lot of stress related genes and genes involved in gene ontological and KEGG pathway were significantly affected due to C. falcatum infection. Quantative real time PCR assays carried out to validate reliability of observed expression patterns in sugarcane in response to C. falcatum infection illustrates first transcriptome wide in planta identification and analysis of TF repertoire in the host pathogen interaction.Conclusion - The results of this study provide a benchmark discovery in finding host targets and provide tissue specific data set of genes that express in response to C. falcatum in sugarcane and also a complete analysis of main group of genes that significantly enriched under this condition. This is the first comprehensive work provides basis for the further studies to dissect role of TFs at molecular level in sugarcane defense to fungal pathogens.

Synergistic Effect of Plant Defense Elicitors and Biocontrol Agents on Induction of Defense Enzymes in Pea against Downy Mildew

Background: Plant defense against the pathogens can be induced by using different defense inducers. Plants can be treated with elicitors for fast and more intense mobilization of defense responses which can enhance the resistance against biotic or abiotic stresses. Methods: The present study has been undertaken to evaluate the synergistic effect of different plant defense inducing chemical (Salicylic acid, Isonicotinic acid, Oxalic acid and Chitosan) and biological (Trichoderma harzianum and Pseudomonas fluorescens) elicitors. Enzyme activity was expressed as the increase in absorbance using spectrophotometer. Result: Among all the treatments the maximum PAL activity (35.58 mg/g of fresh weight) was found in case of oxalic acid but after 48 hrs its activity reduced drastically. Next to oxalic acid Pseudomonas fluorescens (31.38 mg/g of fresh weight), chitosan + Trichoderma harzianum (29.38 mg/g of fresh weight) and chitosan + Pseudomonas fluorescens (27.89 mg/g of fresh weight) showed the maximum enzyme activity. The PPO activity reached the highest at 96 hr after challenge inoculation in case of chitosan + Trichoderma harzianum (9.74 µmol/min/mg/protein) treated plants followed by Trichoderma harzianum (3.53 µmol/min/mg/protein) alone. the maximum PO activity (49.12 µmol/min/mg/protein) was found in case of chitosan + Pseudomonas fluorescens treated plants followed by chitosan (42.48 µmol/min/mg/protein) after 72 hrs. the maximum phenolics (27.53 mg/gm of fresh weight) was found in case of chitosan + Pseudomonas fluorescens after 48 hrs of treatment.

Late Blight Resistance Evaluation and Genome-Wide Assessment of Genetic Diversity in Wild and Cultivated Potato Species

Late blight, caused by the oomycete Phytophthora infestans, is the most devastating disease in potato-producing regions of the world. Cultivation of resistant varieties is the most effective and environmentally friendly way to control potato late blight disease, and identification of germplasms with late blight resistance and clarification their genetic relationship would promote the development of the resistant varieties. In this study, a diverse population of 189 genotypes with potential late blight resistance, consisting of 20 wild species and cultivated Solanum tuberosum Andigenum group and Chilotanum group, was screened for the presence of late blight resistance by performing challenge inoculation with four Phytophthora infestans isolates including one 13_A2 isolate, CN152. Ten elite resources with broad-spectrum resistance and 127 with isolate-specific resistance against P. infestans were identified. To improve the available gene pool for future potato breeding programs, the population was genotyped using 30 simple sequence repeat (SSR) markers covering the entire potato genome. A total of 173 alleles were detected with an average of 5.77 alleles per locus. Structure analysis discriminated the 189 potato genotypes into five populations based on taxonomic classification and genetic origin with some deviations. There was no obvious clustering by country of origin, ploidy level, EBN (endosperm balance number) value, or nuclear clade. Analysis of molecular variance showed 10.08% genetic variation existed among populations. The genetic differentiation (Fst) ranged from 0.0937 to 0.1764, and the nucleotide diversity (π) was 0.2269 across populations with the range from 0.1942 to 0.2489. Further genotyping of 20K SNP array confirmed the classification of SSRs and could uncover the genetic relationships of Solanum germplasms. Our results indicate that there exits abundant genetic variation in wild and cultivated potato germplasms, while the cultivated S. tuberosum Chilotanum group has lower genetic diversity. The phenotypic and genetic information obtained in this study provide a useful guide for hybrid combination and resistance introgression from wild gene pool into cultivated species for cultivar improvement, as well as for germplasm conservation efforts and resistance gene mining.

Effect of Salicylic AcidFormulations on Induced Plant Defense against Cassava Anthracnose Disease

This study was to investigate defense mechanisms on cassava induced by salicylic acid formulation (SA) against anthracnose disease. Our results indicated that the SA could reduce anthracnose severity in cassava plants up to 33.3% under the greenhouse condition. The β-1,3-glucanase and chitinase enzyme activities were significantly increased at 24 hours after inoculation (HAI) and decrease at 48 HAI after Colletotrichum gloeosporioides challenge inoculation, respectively, for cassava treated with SA formulation. Synchrotron radiation–based Fourier-transform infrared microspectroscopy spectra revealed changes of the C=H stretching vibration (3,000-2,800 cm−1), pectin (1,740-1,700 cm−1), amide I protein (1,700-1,600 cm−1), amide II protein (1,600-1,500 cm−1), lignin (1,515 cm−1) as well as mainly C–O–C of polysaccharides (1,300-1,100 cm−1) in the leaf epidermal and mesophyll tissues treated with SA formulations, compared to those treated with fungicide carbendazim and distilled water after the challenged inoculation with C. gloeosporioides. The results indicate that biochemical changes in cassava leaf treated with SA played an important role in the enhancement of structural and chemical defense mechanisms leading to reduced anthracnose severity.

Determinants of Persistent Patterns of Pepino Mosaic Virus Mixed Infections

Pepino mosaic virus (PepMV) has become a pandemic virus in tomato crops, causing important economic losses worldwide. In Spain, isolates of the EU and CH2 strains co-circulate, with PepMV-EU predominantly found in mixed infections. Simultaneous in planta mixed infections result in an asymmetric antagonism against PepMV-CH2, but the outcome of over-infections has never been tested. PepMV-EU and PepMV-CH2 time-lagged inoculations were performed, and viral accumulation was measured 10 days after challenge inoculation. PepMV-EU had a protective effect over PepMV-CH2; in contrast, the accumulation of PepMV-EU increased in plants pre-inoculated with PepMV-CH2 as compared to single infections. We also studied the effect of the type of infection on viral transmission. Independently of the nature of the infection (single or mixed), we observed a strong positive correlation between virus accumulation in the source plant and transmission, excluding mixed infection effects different than modulating viral accumulation. Finally, in order to determine the genetic variability of PepMV strains in single and mixed infections, a 430 nucleotide region was RT-PCR amplified from samples from a serial passages experiment and deep-sequenced. No significant differences were found in the number of nucleotide substitutions between single and mixed infections for PepMV-EU; in contrast, significant differences were found for PepMV-CH2, which was more variable in single than in mixed infections. Comparing PepMV-EU with PepMV-CH2, a higher nucleotide diversity was found for PepMV-CH2. Collectively, our data strongly suggest that PepMV mixed infections can impact the virus epidemiology by modulating in planta virus strain accumulation and diversification.

Vaccination of Algerian Local Rabbits with Precocious Strains of Eimeria magna and Eimeria media

The present study was conducted to assess the safety and the efficacy of a vaccine containing the Algerian precocious strains of Eimeria magna and Eimeria media used separately or together against rabbit coccidiosis. The samples consisted of 56 young rabbits reared in specific pathogen-free conditions. Following the challenge inoculation, statistically significant decreases in oocyst excretion were noticed in the vaccinated rabbits with the precocious strain of Eimeria magna, Eimeria media, and both species leading toa good immune response acquired by the vaccination associated with a good growth rate. Moreover, there was a statistically significant increase in oocyst output following the challenge in all challenged groups. Unlike the vaccinated groups, the challenged groups showed poor weight gains. More than 50% of the young rabbits from all the challenged groups presented diarrhea. Consequently, these precocious strains constitute good candidates for mono or polyvalent anticoccidial vaccines in the future.

Resistance to ‘Candidatus Liberibacter asiaticus,’ the Huanglongbing Associated Bacterium, in Sexually and/or Graft-Compatible Citrus Relatives

Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of genetic resistance to HLB-associated ‘Candidatus Liberibacter asiaticus’ (Las) becomes strategic to warrant crop sustainability, but no resistant Citrus genotypes exist. Some Citrus relatives of the family Rutaceae, subfamily Aurantioideae, were described as full-resistant to Las, but they are phylogenetically far, thus incompatible with Citrus. Partial resistance was indicated for certain cross-compatible types. Moreover, other genotypes from subtribe Citrinae, sexually incompatible but graft-compatible with Citrus, may provide new rootstocks able to restrict bacterial titer in the canopy. Use of seedlings from monoembryonic species and inconsistencies in previous reports likely due to Las recalcitrance encouraged us to evaluate more accurately these Citrus relatives. We tested for Las resistance a diverse collection of graft-compatible Citrinae species using an aggressive and consistent challenge-inoculation and evaluation procedure. Most Citrinae species examined were either susceptible or partially resistant to Las. However, Eremocitrus glauca and Papua/New Guinea Microcitrus species as well as their hybrids and those with Citrus arose here for the first time as full-resistant, opening the way for using these underutilized genotypes as Las resistance sources in breeding programs or attempting using them directly as possible new Las-resistant Citrus rootstocks or interstocks.

A Sec-Dependent Secretory Protein of the Huanglongbing-Associated Pathogen Suppresses Hypersensitive Cell Death in Nicotiana benthamiana

“Candidatus Liberibacter asiaticus” (CLas) is a phloem-restricted Gram-negative bacterium that is the causal agent of citrus huanglongbing (HLB). In this study, we identified a CLas-encoded Sec-dependent secretory protein CLIBASIA_04405 that could contribute to the pathogenicity of this bacterium. The gene expression level of CLIBASIA_04405 was significantly higher in citrus than in psyllids. Transient overexpression of the mature CLIBASIA_04405 protein (m4405) in Nicotiana benthamiana leaves could suppress hypersensitive response (HR)-based cell death and H2O2 accumulation triggered by the mouse BAX and the Phytophthora infestans INF1. An alanine-substitution mutagenesis assay revealed the essential of amino acid clusters EKR45–47 and DE64–65 in cell death suppression. Challenge inoculation of the transgenic N. benthamiana-expressing m4405 with Pseudomonas syringae DC3000ΔhopQ1-1 demonstrated the greatly reduced bacterial proliferation. Remarkably, transcriptome profiling and RT-qPCR analysis disclosed that the gene expression of six small heat shock proteins (sHSPs), a set of plant defense regulators, were significantly elevated in the transgenic m4405 lines compared with those in wild-type N. benthamiana. In addition, the transgenic m4405 lines displayed phenotypes of dwarfism and leaf deformation. Altogether, these data indicated that m4405 was a virulence factor of CLas.