The FBA Motif-Containing Protein NpFBA1 Causes Leaf Curling and Reduces Resistance to Black Shank Disease in Tobacco

Plant leaf morphology has a great impact on plant drought resistance, ornamental research and leaf yield. In this study, we identified a new gene in Nicotiana plumbaginifolia, NpFBA1, that causes leaf curl. The results show that the NpFBA1 protein contains only one unique F-box associated (FBA) domain and does not have an F-box conserved domain. Phylogenetic analysis placed this gene and other Nicotiana FBA genes on a separate branch, and the NpFBA1 protein localized to the nucleus and cytoplasm. The expression of NpFBA1 was induced by black shank pathogen (Phytophthora parasitica var. nicotianae) infection and treatment with salicylic acid (SA) and methyl jasmonate (MeJA). NpFBA1-overexpressing transgenic lines showed leaf curling and aging during the rosette phase. During the bolting period, the leaves were curly and rounded, and the plants were dwarfed. In addition, NpFBA1-overexpressing lines were more susceptible to disease than wild-type (WT) plants. Further studies revealed that overexpression of NpFBA1 significantly downregulated the expression of auxin response factors such as NtARF3 and the lignin synthesis genes NtPAL, NtC4H, NtCAD2, and NtCCR1 in the leaves. In conclusion, NpFBA1 may play a key role in regulating leaf development and the response to pathogen infection.

Multivariate Characterization of Morpho-biometric Traits of Indigenous Helmeted Guinea Fowl (Numida meleagris) in Nigeria

This study was embarked upon to characterise phenotypically helmeted guinea fowls in three agro-ecologies in Nigeria using multivariate approach. Eighteen biometric characters, four morphological indices and eleven qualitative (phaneroptic) traits were investigated in a total of 569 adult birds (158 males and 411 females). Descriptive statistics, non-parametric Kruskal–Wallis H test followed by the Mann–Whitney U test for post hoc, Multiple Correspondence Analysis (MCA), General Linear Model, Canonical Discriminant Analysis, Categorical Principal Component Analysis and Decision Trees were employed to discern the effects of agro-ecological zone and sex on the morphostructural parameters. Agro-ecology had significant effect (P<0.05; P <0.01) on all the colour traits. In general, the most frequently observed colour phenotype of guinea fowl had pearl plumage colour (54.0%), pale red skin colour (94.2%), black shank colour (68.7%), brown eye colour (49.7%), white earlobe colour (54.8%) and brown helmet colour (72.6%). The frequencies of helmet shape and wattle size were significantly influenced (P <0.01) by agro-ecology and sex. Overall, birds from the Southern Guinea Savanna zone had significantly higher values (P <0.05) for most biometric traits compared to their Sudano-Sahelian and Tropical Rainforest counterparts. They were also more compact (120.83±1.61 vs. 113.96±0.97 vs. 111.33±1.19) and had lesser condition index (8.542±0.17 vs. 9.92±0.10 vs. 9.61±0.13) than their counterparts in the two other zones. The interaction between agro-ecology and sex had significant effect (P <0.05) on some quantitative variables. The MCA and discriminant analysis revealed considerable intermingling of the phaneroptic, biometric traits and body indices especially between the Sudano-Sahelian and Tropical Rainforest birds. Inspite of the high level of genetic admixture, the guinea fowl populations could best be distinguished using wing length, body length and eye colour. However, further complementary work on genomics will guide future selection and breeding programmes geared towards improving the productivity, survival and environmental adaptation of indigenous helmeted guinea fowls in the tropics.

Adaptation of Phytophthora nicotianae to multiple sources of partial-resistance in tobacco.

Host resistance is an important tool in the management of black shank disease of tobacco. While race development leads to rapid loss of single-gene resistance, the adaptation by Phytophthora nicotianae to sources of partial resistance from Beinhart 1000, Florida 301, and the Wz gene region introgressed from Nicotiana rustica is poorly characterized. In greenhouse environments, host genotypes with QTLs conferring resistance from multiple sources were initially inoculated with an aggressive isolate of race 0 or race 1 of P. nicotianae. The most aggressive isolate was selected after each of six host generations to inoculate the next generation of plants. The race 0 isolate demonstrated a continuous gradual increase in disease severity and percent root rot on all sources of resistance except the genotype K 326 Wz/--, where a large increase in both was observed between generations two and three. Adaptation by the race 0 isolate on Beinhart 1000 represents the first report of adaptation to this genotype by P. nicotianae. The race 1 isolate did not exhibit significant increases in aggressiveness over generations, but also exhibited a large increase in aggressiveness on K 326 Wz/-- between generations 3 and 4. Molecular characterization of isolates recovered during selection was completed using ddRADseq, but no polymorphisms were associated with the observed changes in aggressiveness. The rapid adaptation to Wz resistance and the gradual adaptation to other QTLs highlights the need to study the nature of Wz resistance and for conducting field studies on efficacy of resistance-gene rotation for disease management.

Hyperspectral reflectance for non-invasive early detection of black shank disease in flue-cured tobacco

Flue-cured tobacco (Nicotiana tabacum L.) is a high value-per-acre crop that is intensively managed to optimise the yield of high-quality cured leaf. A 15-day study assessed the potential of hyperspectral reflectance data for detecting Phytophthora nicotianae (black shank) incidence in flue-cured tobacco. Hyperspectral reflectance data were taken from a commercial flue-cured tobacco field with a progressing black shank infestation. The effort encompassed two key objectives. First, develop hyperspectral indices and/or machine learning classification models capable of detecting Phytophthora nicotianae (black shank) incidence in flue-cured tobacco. Second, evaluate the model’s ability to separate pre-symptomatic plants from healthy plants. Two hyperspectral indices were developed to detect black shank incidence based on differences in the spectral profiles of asymptomatic flue-cured tobacco plants compared to those with black shank symptoms. While one of the indices is a broad-band index and the other uses narrow wavelength values, the statistical difference between the two indices was not significant and both provided an accurate classification of symptomatic plants. Further analysis of the indices showed significant differences between the index values of healthy and symptomatic plants (α = 0.05). In addition, the indices were able to detect black shank symptoms pre-symptomatically (α = 0.09). Subspace linear discriminant analysis, a machine learning classification, was also used for prediction of black shank incidence with up to 85.7% classification accuracy. The implications of using either spectral indices or machine learning for classification for future black shank research are discussed.

A Novel Bio-Fertilizer Produced by Prickly Ash Seeds with Biochar Addition Induces Soil Suppressiveness against Black Shank Disease on Tobacco

A novel bio-fertilizer, produced from prickly ash seeds (PAS), Bacillus subtilis and biochar, was evaluated for its disease-preventing potential on tobacco black shank caused by Phytophthora nicotianae. The results showed that biochar promoted the growth of Tpb55 in PAS and increased the pH of the organic fertilizer. The final concentration of B. subtilis could reach 1.7 × 1010 cfu g−1 in the biological organic fertilizer (PBB) under the optimal medium under conditions of solid-state fermentation. PBB exhibited a strong fumigation effect on P. nicotianae, including inhibiting mycelium growth, reducing the disease severity and decreasing the pathogen population in rhizospheric soil. PBB treatment also could significantly increase the pH of acidified soil and improve soil nutrition content such as available K, alkali hydrolysable N and organic carbon. High-throughput pyrosequencing of 16S and 18S rRNA genes revealed that 4% PBB addition in soil had significant effects on the diversity and richness of fungi but not on that of bacteria. The microbial community structure was also shifted after PBB treatment. Some potentially beneficial microbes such as Bacillus, Mucor, Cunninghamella, Chitinophaga and Phenylobacterium were enriched, while potential pathogen Fusarium was significantly decreased. In conclusion, the agricultural waste PAS combined with biochar can replace soybean as a source for the production of biocontrol B. subtilis Tpb55, and the novel bio-fertilizer could effectively control tobacco black shank by pathogen inhibition, soil nutrient improvement and shifting the rhizomicrobial community.

Whole Genome Sequence of Bacillus velezensis Strain GUMT319: A Potential Biocontrol Agent Against Tobacco Black Shank Disease

Phytophthora nicotianae causes black shank, a serious soil-borne disease, in tobacco. In this study, the Bacillus strain GUMT319 was isolated from the rhizosphere of healthy tobacco plants grown in a field in Guizhou with a high incidence of tobacco black shank. Genome sequencing revealed that GUMT319 contained a single circular chromosome 3,940,023 bp in length, with 4,053 predicted genes and an average GC content of 46.6%. Based on phylogenomic analyses, GUMT319 was designated as Bacillus velezensis. The genome of GUMT319 contained more than 60 genes and 13 gene clusters that have previously been found to be active in antifungal mechanisms, biofilm formation, and chemotaxis motility. Additionally, confocal laser scanning microscopy and scanning electron microscopy showed that GUMT319 formed a spatially organized biofilm in vivo. In addition, lauric acid negatively regulated biofilm formation. This is the first study to report that nicotine in tobacco root exudates was a chemoattractant for biocontrol Bacillus strains. In this study, we identified new interactions between beneficial microorganisms and tobacco roots in the rhizosphere. Moreover, dual culture tests in vitro showed that GUMT319 inhibited the growth of P. nicotianae and also displayed inhibitory effects against eight other plant pathogens, namely, Colletotrichum scovillei, Colletotrichum capsici, Fusarium carminascens, Sclerotinia sclerotiorum, Alternaria alternata, Phomopsis sp., Phyllosticta sorghina, and Exserohilum turcicum. Furthermore, GUMT319 exhibited &gt; 70% control efficiency against tobacco black shank in field experiments conducted in 2018–2020. Thus, GUMT319 was more effective in controlling the incidence of tobacco black shank than other treatments including fungicide application. Overall, these results suggested that GUMT319 (B. velezensis) could be used as a potential biocontrol agent against tobacco black shank.

RNAseq Reveals Differential Gene Expression Contributing to Phytophthora nicotianae Adaptation to Partial Resistance in Tobacco

Phytophthora nicotianae is a devastating oomycete plant pathogen with a wide host range. On tobacco, it causes black shank, a disease that can result in severe economic losses. Deployment of host resistance is one of the most effective means of controlling tobacco black shank, but adaptation to complete and partial resistance by P. nicotianae can limit the long-term effectiveness of the resistance. The molecular basis of adaptation to partial resistance is largely unknown. RNAseq was performed on two isolates of P. nicotianae (adapted to either the susceptible tobacco genotype Hicks or the partially resistant genotype K 326 Wz/Wz) to identify differentially expressed genes (DEGs) during their pathogenic interactions with K 326 Wz/Wz and Hicks. Approximately 69% of the up-regulated DEGs were associated with pathogenicity in the K 326 Wz/Wz-adapted isolate when sampled following infection of its adapted host K 326 Wz/Wz. Thirty-one percent of the up-regulated DEGs were associated with pathogenicity in the Hicks-adapted isolate on K 326 Wz/Wz. A broad spectrum of over-represented gene ontology (GO) terms were assigned to down-regulated genes in the Hicks-adapted isolate. In the host, a series of GO terms involved in nuclear biosynthesis processes were assigned to the down-regulated genes in K 326 Wz/Wz inoculated with K 326 Wz/Wz-adapted isolate. This study enhances our understanding of the molecular mechanisms of P. nicotianae adaptation to partial resistance in tobacco by elucidating how the pathogen recruits pathogenicity-associated genes that impact host biological activities.

Potato virus X-mediated constitutive expression of Plutella xylostella PxSDF2L1 gene in Nicotiana benthamiana confers resistance to Phytophthora parasitica var. nicotianae

Background The Plutella xylostella PxSDF2L1 gene was previously reported to enhance insect resistance to pathogen at high basal transcription rate. PxSDF2L1 shows similitude with the stromal cell-derived factor 2 (SDF2), an ER stress-induced chaperon protein that is highly conserved throughout animals and plants. The precise biological function of SDF2 is not clear, but its expression is required for innate immunity in plants. Here, we investigate whether a continuous expression of PxSDF2L1 in Nicotiana benthamiana can similarly confer resistance to plant pathogen, particularly, the black shank Phytophthora parasitica var. nicotianae. Results The N. benthamiana plants were inoculated with agrobacteria transformed with a PVX-based binary vector carrying the PxSDF2L1 gene; similar agroinoculation experiments with a PVX vector carrying the GFP gene were used for controls. In pot trials, agroinfected N. benthamiana plants constitutively expressing PxSDF2L1 showed a significant reduction of stem disease symptoms caused by the inoculation with P. parasitica, compared with controls. Conclusions We confirm a role of PxSDF2L1 in resistance to black shank, with a potential application to engineering active resistance against this oomycete in the commercial N. tabacum species and propose its evaluation in other crop families and plant pathogens.

Components of aggressiveness in Phytophthora nicotianae during adaptation to multiple sources of partial resistance in tobacco

Black shank is a devastating disease of tobacco caused by Phytophthora nicotianae. Host resistance has been an integral part of black shank management, but after the loss of Php single-gene resistance following its widespread deployment in the 1990s, growers have relied on varieties with varying levels of partial resistance. Partial resistance is effective in suppressing disease, but continued exposure can result in an increase in pathogen aggressiveness that threatens durability of the resistance to P. nicotianae. Aggressiveness components in P. nicotianae were characterized following adaptation on two sources of partial resistance, Fla 301 and the Wz gene from Nicotiana rustica. An aggressive isolate of the two major races of P. nicotianae, race 0 and race 1, were adapted for either ‘one/two’ or ‘five/six’ generations on the two resistance sources, giving four sets of isolates based on race, number of generations of adaptation, and source of resistance. Across the four sets of isolates, adapted isolates infected higher proportions of root tips, produced more sporangia per infected root tip, and caused larger lesions than their respective non-adapted isolates of the same race and from the same resistance source. Adapted isolates also produced more aggressive zoospore progeny than the non-adapted isolates. Adaptation to partial resistance involves multiple aggressiveness components that results in the increased aggressiveness observed for P. nicotianae. These results improve our knowledge on the nature of P. nicotianae adaptation to partial resistance in tobacco and indicate that different resistance sources are likely to select for similar aggressiveness components in the pathogen.