Part II: Impedance-based DNA biosensor for detection of isolated strains of phytopathogen Ralstonia solanacearum.

In Part I, we demonstrated the complete development of a label-free, ultra-low sample volume requiring DNA-based biosensor to detect Ralstonia solanacearum, an aerobic non-spore-forming, Gram-negative, plant pathogenic bacterium, using non-faradaic electrochemical impedance spectroscopy (nf-EIS). We also presented the sensor's sensitivity, specificity, and electrochemical stability. In this article, we highlight the specificity study of the developed DNA-based impedimetric biosensor to detect various strains of R. solanacearum. We have collected seven isolates of R. solanacearum isolated from locally infected host plants (eggplant, potato, tomato, chilli, and ginger) from different parts of Goa, India. The pathogenicity of these isolates was tested on the eggplant, and the pathogen was confirmed by microbiological plating and polymerase chain reaction (PCR). We further report the insight into the DNA hybridization on the surface of Interdigitated Electrodes (IDEs) and the expansion of the Randles model for more accurate analysis. The interpretation of the sensor specificity is clearly demonstrated by the capacitance change observed at the electrode-electrolyte interface.

Part I: Non-faradaic electrochemical impedance-based DNA biosensor for detecting phytopathogen - Ralstonia solanacearum.

Herein, we report for the first time the development of a label-free, non-faradaic, and highly sensitive DNA-based impedimetric sensor using micro-sized gold interdigitated electrodes (IDE) to detect a soil-borne agricultural pathogen Ralstonia solanacearum. A universal 30 oligomer single-stranded DNA (ssDNA) probe lpxC4 having specificity towards R. solanacearum is successfully immobilized on the surface of IDE along with mercaptohexanol. The electrochemical stability of the developed sensor surface is determined using open circuit potential measurements. The DNA probe immobilization protocol is validated using the changes configured on the surface of IDE by contact angle and ATR-FTIR analysis. The DNA target hybridization is detected using non-faradaic electrochemical impedance spectroscopy measurement with an ultra-low sample volume of 10 µL. The non-faradaic approach is verified by studying redox behavior using cyclic voltammetry. We investigate the hybridization of the surface-immobilized label-free probe with the complementary DNA targets obtained from infected eggplant saplings and cross-reactive studies with mismatched DNA strands. Our impedimetric sensor can detect target concentrations as low as 0.1 ng/µL. This standardization and detection of DNA hybridization serves as part I of the work and paves the way for further study in the detection of pathogenic field samples.

Predicting the invasion risk of rugose spiraling whitefly, Aleurodicus rugioperculatus, in India based on CMIP6 projections by MaxEnt.

BACKGROUND: Rugose spiraling whitefly (RSW), Aleurodicus rugioperculatus Martin, is a highly polyphagous invasive pest native to Central America. The occurrence of A. rugioperculatus in the Oriental region was first reported from Pollachi, Tamil Nadu, India in 2017. This pest is widely distributed in India, causing severe economic damage to coconut and other horticultural crops. It is a recent invasion in India and information on its potential distribution is lacking. Thus, in the present study we used the latest Coupled Model Intercomparison Project phase 6 (CMIP6) dataset through Maximum Entropy species distribution modelling (version 3.4.1, MaxEnt) to determine the potential distribution of RSW in present and future climate change scenarios in 2050 and 2070 under Shared Socioeconomic Pathway (SSP) 126 and SSP585 emission scenarios. The performance of the model was evaluated using the area under the curve (AUC), true skill statistics (TSS) and the continuous Boyce index (CBI). RESULTS: The MaxEnt model performed well and predicted the potential distribution of A. rugioperculatus with high-accuracy AUC values of 0.991 and 0.989, TSS values of 0.891 and 0.842, and CBI values of 0.972 and 0.934 for training and testing, respectively. Jackknife analysis revealed that A. rugioperculatus distribution was mostly influenced by temperature-based bioclimatic variables contributing 62.1% of the suitability, with precipitation variables contributing the remainder. The most important bioclimatic variables for RSW distribution were annual mean temperature (Bio 1, 28.9%) followed by mean diurnal range (Bio 2, 19.5%) and annual precipitation (Bio 12, 19.1). Potential suitable areas for RSW establishment were mostly found in all coastal and southern states of India. A. rugioperculatus prefers a warm and humid climate, indicating that the tropics, subtropics and temperate regions are ideal for its spread and invasion. Our results highlighted that the suitable habitat area for A. rugioperculatus is predicted to increase and highest probability of invasion and spread in 2050 and 2070 under future climate change scenarios of SSP126 and SSP585 compared to present climatic conditions. CONCLUSIONS: This is the first study to use the latest CMIP6 models and it predicts the potential distribution of RSW in India under present and future climate change scenarios. The implementation of strict domestic quarantine measures may prevent the spread and damage of RSW to noncoastal regions of India. The results of the current study should help in timely monitoring and surveillance of RSW and to formulate integrated pest management strategies at the national level to restrict its spread, invasion and damage to new areas. © 2022 Society of Chemical Industry.

Corrigendum: Characterization of Fusarium Spp. Inciting Vascular Wilt of Tomato and Its Management by a Chaetomium-Based Biocontrol Consortium.

[This corrects the article DOI: 10.3389/fpls.2021.748013.].

Characterization of Fusarium Spp. Inciting Vascular Wilt of Tomato and Its Management by a Chaetomium-Based Biocontrol Consortium.

Though the vascular wilt of tomato caused by the species of Fusarium is globally reported to be a complex disease in certain countries, for example, India, our studies indicated that the disease is caused by either Fusarium oxysporum f. spp. lycopersici (Fol) or Fusarium solani (FS) with the Fol being widely prevalent. In assessing the genetic diversity of 14 Fol strains representing the four Indian states by the unweighted pair group method with arithmetic averaging using Inter Simple Sequence Repeat (ISSR) amplicons, the strains distinguished themselves into two major clusters showing no correlation with their geographic origin. In pot experiments under polyhouse conditions, the seed dressing and soil application of a talc-based formulation of a biocontrol treatment, TEPF-Sungal-1 (Pseudomonas putida) + S17TH (Trichoderma harzianum) + CG-A (Chaetomium globosum), which inhibited Fol, was equally effective like the cell suspensions and was even better than the fungicidal mixture (copper oxychloride-0.25% + carbendazim-0.1%) in promoting the crop growth (52.3%) and reducing vascular wilt incidence (75%) over the control treatment, despite the challenge of inoculation with a highly pathogenic TOFU-IHBT strain. This was associated with significant expressions of the defense genes, indicating the induction of host resistance by a biocontrol consortium. In field experiments on two locations, the bioconsortium was highly effective in recording maximum mean fruit yields (54.5 and 60%) and a minimum mean vascular wilt incidence (37.5%) in comparison to the untreated control. Thus, Chaetomium-based bioconsortium demonstrated consistency in its performance across the two experiments in 2 years under the two field conditions.

The involvement of the Type Six Secretion System (T6SS) in the virulence of Ralstonia solanacearum on brinjal.

Ralstonia solanacearum is an important soil-borne plant pathogen which causes bacterial wilt in a large number of crops. Bacterial Type Six Secretion System (T6SS) is known to participate in pathogenesis, bacterial interaction and inter-bacterial competition. Contribution of T6SS in the virulence of R. solanacearum on eggplant (Solanum melongena L) is studied. In this study, five T6SS gene (ompA, vgrG3, hcp, tssH and tssM) mutants have been developed by insertional mutagenesis and the virulence of the mutants was evaluated on eggplant. In general, the T6SS mutants showed significant reduction of wilt on eggplant. R. solanacearum mutant of ompA gene significantly reduced the wilt from day five through day eight in petiole inoculation. In soil drench inoculation, R. solanacearum mutant of vgrG3 gene reduced the wilt on eggplant and was significantly different throughout the experimental period. Other mutants, viz., tssH, tssM and hcp, also reduced the wilt during the initial stages of disease development. This is the first report on the role of T6SS genes, ompA, vgrG3, hcp and tssH on virulence of R. solanacearum.

Development of T3SS Mutants (hrpB- and hrcV-) of Ralstonia solanacearum, Evaluation of Virulence Attenuation in Brinjal and Tomato-A Pre-requisite to Validate T3Es of R. solanacearum.

Ralstonia solanacearum is an important plant pathogen which infects a large number of agriculturally important crops. The Type Three Secretion System (T3SS) plays a major role in its pathogenicity by secreting type III effectors (T3Es) which overthrow the host defence mechanism. The secretion of T3Es is transcriptionally regulated by hrpB and its secretion is dependent on the pili formed by hrcV gene. In this study, two T3SS mutants of R. solanacearum strain Rs-09-161 viz. Rs-HrpB- and Rs-HrcV- were developed through insertional mutagenesis. The method of development of insertional mutant is quite simple and reliable. The plasmid integrates through homologous recombination and in vitro studies have proved that the integration was stable for several generations. The mutants are non-pathogenic on its highly susceptible hosts, brinjal and tomato inoculated by soil drench method and by petiole inoculation directly into the vascular system. Further it was observed that the colonisation ability of the mutants was also highly reduced in the susceptible host. These mutants will be useful in validating putative T3E through translocation studies.

Identification of virulence factors and type III effectors of phylotype I, Indian Ralstonia solanacearum strains Rs-09-161 and Rs-10-244.

Ralstonia solanacearum is a well-known phytopathogen causing bacterial wilt in a large number of agriculturally important crops. The pathogenicity of R. solanacearum is expressed due to the presence of various virulence factors and effector proteins. In this study, various virulence factors and type III effector proteins of R. solanacearum that are present in the strains Rs-09-161 and Rs-10-244 were identified through bioinformatics approach and compared with other reference strains. R. solanacearum strains, Rs-09-161 and Rs-10-244 belong to the phylotype I, biovar3, and are the only sequenced strains from India infecting solanaceous vegetables. Similarity matrix obtained by comparing the sequences of virulence genes of Rs-09-161 and Rs-10-244 with other reference strains indicated that Rs-09-161 and Rs-10-244 share more than 99% similarity between them and are closely related to GMI1000. The virulence factors in R. solanacearum appear to be highly conserved in the R. solanacearum species complex. Rs-09-161 has 72 type III effectors whereas Rs-10-244 has 77. Comparison of the complete genes of type III effectors of Rs-09-161,Rs-10-244 andGMI1000 revealed the presence of 60 common effectors within them. Further,Rs-09-161 has two unique effectors and Rs-10-244 has four unique effectors. Phylogenetic trees of RipA, RipG, RipH and RipS effector sequences resulted in the grouping of the isolates based on their phylotypes. Group 1 consists of strains that belong to phylotype I including Rs-09-161 and Rs-10-244. Phylotype III strain CMR15 forms a group closely associated with phylotype I. The strains belonging to phylotypes II and IV have separated to form two different groups.

Diversity, biocontrol, and plant growth promoting abilities of xylem residing bacteria from solanaceous crops.

Eggplant (Solanum melongena L.) is one of the solanaceous crops of economic and cultural importance and is widely cultivated in the state of Goa, India. Eggplant cultivation is severely affected by bacterial wilt caused by Ralstonia solanacearum that colonizes the xylem tissue. In this study, 167 bacteria were isolated from the xylem of healthy eggplant, chilli, and Solanum torvum Sw. by vacuum infiltration and maceration. Amplified rDNA restriction analysis (ARDRA) grouped these xylem residing bacteria (XRB) into 38 haplotypes. Twenty-eight strains inhibited growth of R. solanacearum and produced volatile and diffusible antagonistic compounds and plant growth promoting substances in vitro. Antagonistic strains XB86, XB169, XB177, and XB200 recorded a biocontrol efficacy greater than 85% against BW and exhibited 12%-22 % increase in shoot length in eggplant in the greenhouse screening. 16S rRNA based identification revealed the presence of 23 different bacterial genera. XRB with high biocontrol and plant growth promoting activities were identified as strains of Staphylococcus sp., Bacillus sp., Streptomyces sp., Enterobacter sp., and Agrobacterium sp. This study is the first report on identity of bacteria from the xylem of solanaceous crops having traits useful in cultivation of eggplant.

Genome Sequencing of Ralstonia solanacearum Biovar 3, Phylotype I, Strains Rs-09-161 and Rs-10-244, Isolated from Eggplant and Chili in India.

Ralstonia solanacearum Indian strains Rs-09-161 and Rs-10-244 were isolated from the coastal region of Goa and from the Andaman Islands. We report the draft genome sequences of these representative isolates infecting solanaceous vegetables in India.