Plants

The devastating citrus disease, Huanglongbing (HLB), is associated with ‘Candidatus Liberibacter sp.’ and transmitted by citrus psyllids. Unfortunately, HLB has no known sustainable cure yet. Herein, we proposed γ-aminobutyric acid (GABA) as a potential eco-friendly therapeutic solution to HLB. Herein, we used GC/MS-based targeted metabolomics combined with gene expression to investigate the role of GABA in citrus response against HLB and to better understand its relationship(s) with different phytohormones. GABA supplementation via root drench boosts the accumulation of endogenous GABA in the leaves of both healthy and ‘Ca. L. asiaticus’-infected trees. GABA accumulation benefits the activation of a multi-layered defensive system via modulating the phytohormone levels and regulating the expression of their biosynthesis genes and some pathogenesis-related proteins (PRs) in both healthy and ‘Ca. L. asiaticus’-infected plants. Moreover, our findings showed that GABA application stimulates auxin biosynthesis in ‘Ca. L. asiaticus’-infected plants via the activation of the indole-3-pyruvate (I3PA) pathway, not via the tryptamine (TAM)-dependent pathway, to enhance the growth of HLB-affected trees. Likewise, GABA accumulation was associated with the upregulation of SA biosynthesis genes, particularly the PAL-dependent route, resulting in higher SA levels that activated CsPR1, CsPR2, CsPR5, and CsWRKY70, which are prominent to activation of the SA-mediated pathway. Additionally, higher GABA levels were correlated with an enhanced JA profile and linked with both CsPR3 and CsPR4, which activates the JA-mediated pathway. Collectively, our findings suggest that exogenous GABA application might be a promising alternative and eco-friendly strategy that helps citrus trees battle HLB.

Early detection and prompt response are key factors in the eradication of ‘huanglongbing’ (HLB) in California. Currently, qPCR testing of leaf tissue guides the removal of infected trees. However, because of the uneven distribution of ‘Candidatus Liberibacter asiaticus’ (CLas) in an infected tree and asymptomatic infection, selecting the best leaves to sample, from a mature tree with more than 200,000 estimated leaves, is a major hurdle for timely detection. The goal of this study was to address this issue by testing alternative tissues that might improve the CLas detection rate. Using two years of field data, old and young leaves, peduncle bark of fruit, and feeder roots were evaluated for the presence of CLas. Quadrant-peduncle (Q-P) tissue sampling consistently resulted in better CLas detection than any other tissue type. Q-P samples had a 30% higher qPCR positivity rate than quadrant-leaf (Q-L) samples. No significant seasonal patterns were observed. Roots and single peduncles had similar detection rates; both were higher than single leaves or Q-L samples. If symptoms were used to guide sampling, 30% of infected trees would have been missed. Taken together, these results suggest that Q-P tissue sampling is the optimal choice for improved CLas detection under California growing conditions.

“Bois noir” disease associated with ‘Candidatus Phytoplasma solani’ seriously compromises the production and survival of grapevines (Vitis vinifera L.) in Europe. Understanding the plant response to phytoplasmas should help to improve disease control strategies. Using a combined metabolomic and transcriptomic analysis, this work, therefore, investigated the phytoplasma–grapevine interaction in red cultivar Sangiovese in a vineyard over four seasonal growth stages (from late spring to late summer), comparing leaves from healthy and infected grapevines (symptomatic and symptomless). We found an accumulation of both conjugate and free salicylic acids (SAs) in the leaves of ‘Ca. P. solani’-positive plants from early stages of infection, when plants are still asymptomatic. A strong accumulation of gentisic acid (GA) associated with symptoms progression was found for the first time. A detailed analysis of phenylpropanoids revealed a significant accumulation of hydroxycinnamic acids, flavonols, flavan 3-ols, and anthocyanin cyanidin 3-O-glucoside, which are extensively studied due to their involvement in the plant response to various pathogens. Metabolomic data corroborated by gene expression analysis indicated that phenylpropanoid biosynthetic and salicylic acid-responsive genes were upregulated in ‘Ca. P. solani-positive plants compared to -negative ones during the observed period.

Huanglongbing (HLB) is one of the most destructive diseases threatening citriculture worldwide. This disease has been associated with α-proteobacteria species, namely Candidatus Liberibacter. Due to the unculturable nature of the causal agent, it has been difficult to mitigate the disease, and nowadays a cure is not available. MicroRNAs (miRNAs) are key regulators of gene expression, playing an essential role in abiotic and biotic stress in plants including antibacterial responses. However, knowledge derived from non-model systems including Candidatus Liberibacter asiaticus (CLas)-citrus pathosystem remains largely unknown. In this study, small RNA profiles from Mexican lime (Citrus aurantifolia) plants infected with CLas at asymptomatic and symptomatic stages were generated by sRNA-Seq, and miRNAs were obtained with ShortStack software. A total of 46 miRNAs, including 29 known miRNAs and 17 novel miRNAs, were identified in Mexican lime. Among them, six miRNAs were deregulated in the asymptomatic stage, highlighting the up regulation of two new miRNAs. Meanwhile, eight miRNAs were differentially expressed in the symptomatic stage of the disease. The target genes of miRNAs were related to protein modification, transcription factors, and enzyme-coding genes. Our results provide new insights into miRNA-mediated regulation in C. aurantifolia in response to CLas infection. This information will be useful to understand molecular mechanisms behind the defense and pathogenesis of HLB.

Citrus huanglongbing (HLB) disease associated with the ‘Candidatus Liberibacter asiaticus’ (CLas) bacterium has caused significant financial damage to many citrus industries. Large-scale pathogen surveys are routinely conducted in California to detect CLas early in the disease cycle by lab-based qPCR assays. We have developed an improved reference gene for the sensitive detection of CLas from plants in diagnostic duplex qPCR and analytical digital droplet PCR (ddPCR) assays. The mitochondrial cytochrome oxidase gene (COX), widely used as a reference, is not ideal because its high copy number can inhibit amplification of small quantities of target genes. In ddPCRs, oversaturation of droplets complicates data normalization and quantification. The variable copy numbers of COX gene in metabolically active young tissue, greenhouse plants, and citrus relatives suggest the need for a non-variable, nuclear, low copy, universal reference gene for analysis of HLB hosts. The single-copy nuclear gene, malate dehydrogenase (MDH), developed here as a reference gene, is amenable to data normalization, suitable for duplex qPCR and ddPCR assays. The sequence of MDH fragment selected is conserved in most HLB hosts in the taxonomic group Aurantioideae. This study emphasizes the need to develop standard guidelines for reference genes in DNA-based PCR assays.

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.

Understanding temporal biological phenomena is a challenging task that can be approached using network analysis. Here, we explored whether network reconstruction can be used to better understand the temporal dynamics of bois noir, which is associated with ‘Candidatus Phytoplasma solani’, and is one of the most widespread phytoplasma diseases of grapevine in Europe. We proposed a methodology that explores the temporal network dynamics at the community level, i.e., densely connected subnetworks. The methodology offers both insights into the functional dynamics via enrichment analysis at the community level, and analyses of the community dissipation, as a measure that accounts for community degradation. We validated this methodology with cases on experimental temporal expression data of uninfected grapevines and grapevines infected with ‘Ca. P. solani’. These data confirm some known gene communities involved in this infection. They also reveal several new gene communities and their potential regulatory networks that have not been linked to ‘Ca. P. solani’ to date. To confirm the capabilities of the proposed method, selected predictions were empirically evaluated.

The present study examined the effects of Candidatus Phytoplasma solani infection on antioxidative metabolism in leaves and roots of carrot (Daucus carota L.). Disease symptoms appeared at the end of June in the form of the chlorosis on some of the leaves, which became intensely red one week later, while the previously healthy leaves from the same branch becme chlorotic. A few days later, all leaves from the infected leaf branch were intensely red. Infected plants also had slower growth compared to the healthy ones with fewer leaf branches developed. The roots of infected plants were less developed, seared, or gummy with or without brown-colored root hair. The presence of the pathogen was detected by sequencing the 16S rRNA. National Center for Biotechnology Information (NCBI) BLAST analyses of the obtained sequence revealed 100% identity of tested strain with deposited Ca. Phytoplasma solani strains from various countries and hosts, all belonging to the “stolbur” group (16SrXII-A). Identity of 99.74% was found when the tested Serbian strain (MF503627) was compared with the reference stolbur strain STOL11 (AF248959). The oxidative damage of membranes in carrot cells was accompanied by a decrease in the content of photosynthetic pigments. Furthermore, for the determination of specific scavenging properties of the extracts, in vitro antioxidant assay was performed. In phytoplasma-infected carrot leaves, there was a greater reduction in the level of glutathione content (GSH); however; flavonoids and anthocyanidins seem to be responsible for the accompanied increased antioxidative capacity against hydroxyl radical and hydrogen peroxide.

The psyllids Cacopsylla melanoneura and Cacopsylla picta reproduce on apple (Malus × domestica) and transmit the bacterium ‘Candidatus Phytoplasma mali’, the causative agent of apple proliferation. Adult psyllids were collected by the beating-tray method from lower and upper parts of the apple tree canopy in the morning and in the afternoon. There was a trend of catching more emigrant adults of C.melanoneura in the morning and in the lower part of the canopy. For C.melanoneura remigrants, no differences were observed. The findings regarding the distribution of adults were reflected by the number of nymphs collected by wash-down sampling. The density of C.picta was too low for a statistical analysis. The vector monitoring and how it is commonly performed, is suitable for estimating densities of C.melanoneura. Nevertheless, above a certain temperature threshold, prediction of C.melanoneura density might be skewed. No evidence was found that other relatively abundant psyllid species in the orchard, viz. Baeopelma colorata, Cacopsylla breviantennata, Cacopsylla brunneipennis, Cacopsylla pruni and Trioza urticae, were involved in ‘Candidatus Phytoplasma mali’ transmission. The results of our study contribute to an advanced understanding of insect vector behavior and thus have a practical impact for an improved field monitoring.

The bacterial pathogen ‘Candidatus Liberibacter solanacearum’ (Lso) is transmitted by the tomato potato psyllid (TPP), Bactericera cockerelli, to solanaceous crops. In the present study, the changes in metabolic profiles of insect-susceptible (cv CastleMart) and resistant (RIL LA3952) tomato plants in response to TPP vectoring Lso or not, were examined after 48 h post infestation. Non-volatile and volatile metabolites were identified and quantified using headspace solid-phase microextraction equipped with a gas chromatograph-mass spectrometry (HS-SPME/GC-MS) and ultra-high pressure liquid chromatography coupled to electrospray quadrupole time-of-flight mass spectrometry (UPLC/ESI-HR-QTOFMS), respectively. Partial least squares-discriminant analysis (PLS-DA) was used to define the major uncorrelated metabolite components assuming the treatments as the correlated predictors. Metabolic changes in various classes of metabolites, including volatiles, hormones, and phenolics, were observed in resistant and susceptible plants in response to the insects carrying the pathogen or not. The results suggest the involvement of differentially regulated and, in some cases, implicates antagonistic metabolites in plant defensive signaling. Upon validation, the identified metabolites could be used as markers to screen and select breeding lines with enhanced resistance to reduce economic losses due to the TPP-Lso vector-pathogen complex in Solanaceous crops.