Trunk Injection of Citrus Trees with a Polymeric Nanobactericide Reduces Huanglongbing Severity Caused by Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) is a disease caused by the phloem- limited Candidatus Liberibacter asiaticus (CLas) that affects the citrus industry worldwide. To date, only indirect strategies have been implemented to eradicate HLB. Included among these is the population control of the psyllid vector (Diaphorina citri), which usually provides inconsistent results. Even though strategies for direct CLas suppression seem a priori more promising, only a handful of reports have been focused on a confrontation of the pathogen. Recent developments in polymer chemistry have allowed the design of polycationic self-assembled block copolymers with outstanding antibacterial capabilities. Here, we report the use of polymeric nano-sized bactericide particles (PNB) to control CLas directly in the phloem vasculature. The field experiments were performed in Rioverde, San Luis Potosí, and is one of the most important citrusproducing regions in Mexico. An average 52% reduction in the bacterial population was produced when PNB was injected directly into the trunk of 20 infected trees, although, in some cases, reduction levels reached 97%. These results position PNB as a novel and promising nanotechnological tool for citrus crop protection against CLas and other related pathogens.

Enterobacter sp. DBA51 produces ACC deaminase and promotes the growth of tomato (Solanum lycopersicum L.) and tobacco (Nicotiana tabacum L.) plants under greenhouse condition.

Bacterial isolated from rhizospheric soil associated with the semi-desertic plant Coronilla juncea L. were screened for 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity, a common trait for plant-growth-promoting rhizobacteria (PGPR). Among bacterial isolates, strain DBA51 showed phosphate solubilizing index (PSI), producing indole acetic acid (IAA), and with the hemolysis-negative test. Sequencing and analysis of the 16S rDNA gene identified DBA51 as Enterobacter. DBA51 did not show antagonistic activity in vitro against bacterial (Clavibacter michiganensis, Pseudomonas syringae pv. tomato DC3000 and Pectobacterium cacticidum FHLGJ22) and fungal phytopathogens (Alternaria sp., Fusarium oxysporum fsp. lycopersici, Fusarium oxysporum fsp. cubense M5, and Rhizoctonia sp.). Root inoculations with DBA51 in tomato (Solanum lycopersicum L.) and tobacco (Nicotiana tabacum L.) plants were performed under greenhouse conditions. Plant height (20 %) and root biomass (40 %) were significantly enhanced in tomato plants inoculated with DBA51 compared to non-inoculated plants, although for tobacco plants, only root biomass (27 %) showed significant differences with DBA51. In addition, physiological parameters such as photosynthetic rate (µmol CO2 m-2 s-1), stomatal conductance (mol H2O m-2 s-1), and transpiration rate (mmol H2O m-2 s-1) were also evaluated, and no differences were detected between DBA51-inoculated and control treatment in tomato and tobacco leaves. The observed results indicate that the DBA51 strain could be used as a biofertilizer to improve yields of horticultural crops.

Tomato Varieties Influence the Performance of Tamarixia triozae (Hymenoptera: Eulophidae) on Bactericera cockerelli (Hemiptera: Triozidae) Nymphs.

The potato/tomato psyllid Bactericera cockerelli is the Candidatus Liberibacter solanacearum bacterium vector that causes diseases in Solanaceae crops. Pest control is based on synthetic chemical insecticides, plant extracts, and natural enemies such as parasitoids. Tamarixia triozae feeds on nymphs of B. cockerelli, reaching up to 95% parasitism. This work aimed to evaluate the parasitic performance of T. triozae on tomato leaves with B. cockerelli N3 nymphs, using two domesticated (Floradade and Micro-Tom) and one Wild tomato variety. Several assays were completed to identify the parasitoid attraction toward un-infested plants (healthy) and infested plants (damaged) of three varieties. Parasitism preference and "Y" tube olfactometer tests were performed, respectively. The parasitism of Tamarixia triozae showed a preference toward plants of the Floradade variety by 44% compared with the other two varieties (p = 0.0003). T. triozae was more attracted to damaged plants of the Wild variety (p = 0.0523). Healthy plants of Floradade and Micro-Tom varieties attracted a higher proportion of parasitoids, except in the Wild variety, where T. triozae was more attracted to damaged plants. Taken together, the results of this study show that the domestication degree in tomato plants positively influenced the interactions between tomato plants and the parasitoid, T. triozae.

Exogenous Application of Polycationic Nanobactericide on Tomato Plants Reduces the Candidatus Liberibacter Solanacearum Infection.

Candidatus Liberibacter solanacearum (CaLso) is associated with diseases in tomato crops and transmitted by the tomato psyllid Bactericera cockerelli. A polymeric water-dispersible nanobactericide (PNB) was evaluated against CaLso as a different alternative. PNB is a well-defined polycationic diblock copolymer designed to permeate into the vascular system of plants. Its assessment under greenhouse conditions was carried out with tomato plants previously infected with CaLso. Using a concentration as low as 1.0 mg L-1, a small but significant reduction in the bacterial load was observed by real-time qPCR. Thus, to achieve an ecologically friendly dosage and set an optimum treatment protocol, we performed experiments to determine the effective concentration of PNB to reduce ~65% of the initial bacterial load. In a first bioassay, a 40- or 70-fold increase was used to reach that objective. At this concentration level, other bioassays were explored to determine the effect as a function of time. Surprisingly, a real reduction in the symptoms was observed after three weeks, and there was a significant decrease in the bacterial load level (~98%) compared to the untreated control plants. During this period, flowering and formation of tomato fruits were observed in plants treated with PNB.

Fluorescence detection of pyrene-stained Bacillus subtilis LPM1 rhizobacteria from colonized patterns of tomato roots.

A series of water soluble 8-alcoxypyrene-1,3,6-trisulfonic sodium salts bearing different alcoxy lateral chains and functional end groups was synthesized and the molecular structure was corroborated by nuclear magnetic resonance spectroscopy. The photophysical properties in water analyzed by UV-Vis and static and dynamic fluorescence revealed that all of the pigments emit in the blue region at a maximal wavelength of 436 nm and with fluorescence lifetimes in the range of ns. Among them, sodium 8-((10-carboxydecyl) oxy) pyrene-1,3,6-trisulfonate M1 exhibits a high fluorescence quantum yield (φ = 80%) and a good interaction with B. subtilis LPM1 rhizobacteria; this has been demonstrated through in vitro staining assays. Tomato plants (Solanum lycopersicon cv. Micro-Tom) increased the release of root exudates, mainly malic and fumaric acids, after 12 h of treatment with benzothiadiazole (BTH) as a foliar elicitor. However, the chemotaxis analysis demonstrated that malic acid is the most powerful chemoattractant of the rhizobacteria Bacillus subtilis LPM1: in agar plates, a major growth (60 mm) was found for a concentration of 100 mM, while in capillary tubes, the earliest response was at 30 min with 3.3 × 108 CFU mL-1. The confocal microscopic analysis carried out on the tomato roots of the pyrene stained B. subtilis LPM1 revealed that this bacterium mainly colonizes the epidermal zones, i.e. the junctions to primary roots, lateral roots and root hairs, meaning that these root hair sections are the highest colonisable sites involved in the biosynthesis of exudates. This fluorescent pyrene marker M1 represents a valuable tool to evaluate B. subtilis-plant interactions in an easy and quick test in both in vitro and in vivo tomato crops.

Effects of Bactericera cockerelli Herbivory on Volatile Emissions of Three Varieties of Solanum lycopersicum.

Domesticated tomato (Solanum lycopersicum L.) crops have presented an increased susceptibility to pests under field and greenhouse conditions. Among these pests is tomato/potato psyllid, Bactericera cockerelli Sulc (Hemiptera: Triozidae), a major pest in solanaceous crops. In this study, we evaluated volatile organic compound (VOC) emissions from the headspace in three healthy varieties of tomato plants (Floradade, Micro-Tom and wild) under greenhouse conditions using solid-phase microextraction and gas chromatography-mass spectrometry (SPME/GC-MS). Later, independent bioassays were performed to evaluate VOC emissions with three varieties infested with nymphs of B. cockerelli. The results in healthy plants showed markedly different VOC profiles in each variety (14 compounds for wild, 17 for Floradade and 4 for Micro-Tom). Plants infested with nymphs showed changes in VOC emissions distinctly in Floradade and wild varieties. We suggest that these qualitative differences in VOC profiles by the degree of domestication could explain the preferences of B. cockerelli.

Critical Minireview: The Fate of tRNACys during Oxidative Stress in Bacillus subtilis.

Oxidative stress occurs when cells are exposed to elevated levels of reactive oxygen species that can damage biological molecules. One bacterial response to oxidative stress involves disulfide bond formation either between protein thiols or between protein thiols and low-molecular-weight (LMW) thiols. Bacillithiol was recently identified as a major low-molecular-weight thiol in Bacillus subtilis and related Firmicutes. Four genes (bshA, bshB1, bshB2, and bshC) are involved in bacillithiol biosynthesis. The bshA and bshB1 genes are part of a seven-gene operon (ypjD), which includes the essential gene cca, encoding CCA-tRNA nucleotidyltransferase. The inclusion of cca in the operon containing bacillithiol biosynthetic genes suggests that the integrity of the 3' terminus of tRNAs may also be important in oxidative stress. The addition of the 3' terminal CCA sequence by CCA-tRNA nucleotidyltransferase to give rise to a mature tRNA and functional molecules ready for aminoacylation plays an essential role during translation and expression of the genetic code. Any defects in these processes, such as the accumulation of shorter and defective tRNAs under oxidative stress, might exert a deleterious effect on cells. This review summarizes the physiological link between tRNACys regulation and oxidative stress in Bacillus.

Transformed tobacco (Nicotiana tabacum) plants over-expressing a peroxisome proliferator-activated receptor gene from Xenopus laevis (xPPARα) show increased susceptibility to infection by virulent Pseudomonas syringae pathogens.

Transgenic tobacco plants capable of over-expressing Xenopus PPARα (xPPARα), a transcription factor known to be required for peroxisome proliferation in animals, were recently generated. These plants (herewith referred to as PPAR-OE) were found to have increased peroxisome abundance, higher peroxisomal acyl-CoA oxidase and catalase activity and modified fatty acid metabolism. Further characterization of PPAR-OE plants revealed a higher susceptibility to virulent and a partial loss of resistance to avirulent Pseudomonas syringae pathogens, whereas the basal resistance response remained unaffected. Biochemical- and defense-related gene expression analyses showed that increased susceptibility to bacterial invasion coincided with the generalized reduction in H(2)O(2) and salicylic acid (SA) levels observed within the first 24 h of bacterial contact. Decreased H(2)O(2) levels were correlated with modified activity levels of catalase and other antioxidant enzymes. A correspondence between a rapid (within 1-24 hpi; ACCO and AOC) and sustained increase (up to 6 days pi; ACCO) in the expression levels of ethylene (ACCO) and jasmonic acid (AOC) biosynthetic genes and a higher susceptibility to virulent bacterial invasion was also observed in PPAR-OE plants. Conversely, no apparent differences in the short- and/or long-term expression levels of markers for the hypersensitive-response, oxidative burst and systemic-acquired resistance were observed between wild type and PPAR-OE plants. The results suggest that peroxisome proliferation could lead to increased susceptibility to bacterial pathogens in tobacco by altering the redox balance of the plant and the expression pattern of key defense signaling pathway genes.

Inoculation of tomato plants (Solanum lycopersicum) with growth-promoting Bacillus subtilis retards whitefly Bemisia tabaci development.

Root inoculation of tomato (Solanum lycopersicum) plants with a Bacillus subtilis strain BEB-DN (BsDN) isolated from the rhizosphere of cultivated potato plants was able to promote growth and to generate an induced systemic resistance (ISR) response against virus-free Bemisia tabaci. Growth promotion was evident 3 weeks after inoculation. No changes in oviposition density, preference and nymphal number in the early stages of B. tabaci development were observed between BsDN-treated plants and control plants inoculated with a non-growth promoting Bs strain (PY-79), growth medium or water. However, a long-term ISR response was manifested by a significantly reduced number of B. tabaci pupae developing into adults in BsDN-treated plants. The observed resistance response appeared to be a combination of jasmonic acid (JA) dependent and JA-independent responses, since the BsDN-related retardation effect on B. tabaci development was still effective in the highly susceptible spr2 tomato mutants with an impaired capacity for JA biosynthesis. A screening of 244 genes, 169 of which were previously obtained from subtractive-suppressive-hybridization libraries generated from B. tabaci-infested plants suggested that the BsDN JA-dependent ISR depended on an anti-nutritive effect produced by the simultaneous expression of genes coding principally for proteases and proteinase inhibitors, whereas the JA-independent ISR observed in the spr2 background curiously involved the up-regulation of several photosynthetic genes, key components of the phenyl-propanoid and terpenoid biosynthetic pathways and of the Hsp90 chaperonin, which probably mediated pest resistance response(s), in addition to the down-regulation of pathogenesis and hypersensitive response genes.

Differential gene expression in whitefly Bemisia tabaci-infested tomato (Solanum lycopersicum) plants at progressing developmental stages of the insect's life cycle.

A suppression-subtractive-hybridization (SSH) strategy was used to identify genes whose expression was modified in response to virus-free whitefly Bemisia tabaci (Bt, biotype A) infestation in tomato (Solanum lycopersicum) plants. Thus, forward and reverse SSH gene libraries were generated at four points in the whitefly's life cycle, namely at (1) 2 days (adult feeding and oviposition: phase I); (2) 7 days (mobile crawler stage: phase II); (3) 12 days (second to third instar nymphal transition: phase III) and (4) 18 days (fourth instar nymphal stage: phase IV). The 169 genes with altered expression (up and downregulated) that were identified in the eight generated SSH libraries, together with 75 additional genes that were selected on the basis of their involvement in resistance responses against phytofagous insects and pathogens, were printed on a Nexterion(®) Slide MPX 16 to monitor their pattern of expression at the above phases. The results indicated that Bt infestation in tomato led to distinctive phase-specific expression/repression patterns of several genes associated predominantly with photosynthesis, senescence, secondary metabolism and (a)biotic stress. Most of the gene expression modifications were detected in phase III, coinciding with intense larval feeding, whereas fewer changes were detected in phases I and IV. These results complement previously reported gene expression profiles in Bt-infested tomato and Arabidopisis, and support and expand the opinion that Bt infestation leads to the downregulation of specific defense responses in addition to those controlled by jasmonic acid.