Citrus Huanglongbing (HLB) or citrus greening, is the most destructive disease for citrus worldwide. It is caused by the psyllid-transmitted, phloem-limited bacteria "
Liberibacter asiaticus" (
Las). ...To date, there are still no effective practical strategies for curing citrus HLB. Understanding the mechanisms against
Las can contribute to the development of effective approaches for combatting HLB. However, the unculturable nature of
Las has hindered elucidating mechanisms against
Las. In this review, we summarize the main aspects that contribute to the understanding about the mechanisms against
Las, including (1)
Las virulence targets, focusing on inhibition of virulence genes; (2) activation of citrus host defense genes and metabolites of HLB-tolerant citrus triggered by
Las, and by agents; and (3) we also review the role of citrus microbiome in combatting
Las. Finally, we discuss novel strategies to continue studying mechanisms against
Las and the relationship of above aspects.
Citrus is a globally important, perennial fruit crop whose rhizosphere microbiome is thought to play an important role in promoting citrus growth and health. Here, we report a comprehensive analysis ...of the structural and functional composition of the citrus rhizosphere microbiome. We use both amplicon and deep shotgun metagenomic sequencing of bulk soil and rhizosphere samples collected across distinct biogeographical regions from six continents. Predominant taxa include Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes. The core citrus rhizosphere microbiome comprises Pseudomonas, Agrobacterium, Cupriavidus, Bradyrhizobium, Rhizobium, Mesorhizobium, Burkholderia, Cellvibrio, Sphingomonas, Variovorax and Paraburkholderia, some of which are potential plant beneficial microbes. We also identify over-represented microbial functional traits mediating plant-microbe and microbe-microbe interactions, nutrition acquisition and plant growth promotion in citrus rhizosphere. The results provide valuable information to guide microbial isolation and culturing and, potentially, to harness the power of the microbiome to improve plant production and health.
The citrus industry is facing an unprecedented challenge from Huanglongbing (HLB). All cultivars can be affected by the HLB-associated bacterium 'Candidatus Liberibacter asiaticus' (CLas) and there ...is no known resistance. Insight into HLB pathogenesis is urgently needed in order to develop effective management strategies. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all CLas isolates, as a molecular probe to understand CLas virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in CLas-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1-expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants.
Huanglongbing (HLB) is an important citrus disease associated with the phloem-limited, uncultured bacterium ‘Candidatus Liberibacter asiaticus’(CLas). Effective treatments against CLas have to be ...validated in the field, however, methods for the field assessment of treatment effectiveness are time-consuming, in part because DNA-based assays, including quantitative PCR (qPCR), cannot differentiate between live and dead bacterial DNA. The aim of this study was to develop a method for rapid the evaluation of HLB therapies in field experiments. To this aim, a DNA extraction method from citrus leaf tissues with propidum monoazide (PMA), a dye that binds covalently to dsDNA making it unavailable for amplification in subsequent qPCR reactions, was optimized. The results indicated that the efficacy of PMA-qPCR was highly dependent on the primer set used. Primers targeting the 16S region of CLas showed a clear distinction between qPCR from PMA-treated and non-treated samples, while the RNR and LJ900 primers did not show significant differences between the DNA extraction methods. The PMA-qPCR viability analysis of CLas from citrus cuttings treated with different ampicillin (Amp) concentrations showed that all concentrations reduced CLas titers significantly starting 4 days after the initial treatment, unlike the water treatment, which did not show any change. This method was used for assessing the antibacterial activity of Amp, Streptomycin, Oxytetracycline (OTC), and a water control in field tests. The PMA-qPCR results indicated that Amp and OTC displayed significant antibacterial activity against CLas by 8 days post-injection, which was not detected in the non-PMA qPCR analysis. This method could allow the rapid validation of treatments against CLas in field experiments and facilitate the implementation of effective management strategies against HLB.
Abstract
Hydrochemical characteristics of irrigation water and their spatiotemporal variations can provide critical information for ensuring healthy crop growth and determining the best water ...management practices. The Lower Rio Grande Valley (LRGV) is heavily dependent upon ditch irrigation to deliver water from the Rio Grande River to support its staple crop production. To date, no studies have been conducted to quantify the water quality and its variations along the distribution system. This research measured water quality parameters at seven sites in LRGV irrigation water in 2021. Chemical indices including salinity hazard (SH), sodium adsorption ratio (SAR), sodium percentage (Na%), residual sodium carbonate (RSC), magnesium hazard (MH), Kelly's Ratio (KR), and permeability index (PI) were calculated. Classification diagrams were prepared. Results revealed the locations that had doubtful water for irrigation use and more problematic water quality index levels. June and August had the highest index levels, which may have been attributable to the large rainfall events in May and July. The SH, Na%, KR, and MH indices exceeded recommended levels. ANOVA analyses showed significant temporal variations in SAR, RSC, MH, KR, and PI. These findings indicate the importance of incorporating water quality spatiotemporal variation information in routine irrigation planning and management.
Root endophytes have been shown to influence plant growth and plant responses to pathogens. Phytophthora spp. are important pathogens of citrus that cause foot rot and fibrous root decline. We ...hypothesize that foot rot infection by Phytophthora spp. can result in changes of the endophytic microbial community in citrus roots. Therefore, the endophytic (including bacterial and fungal) microbiome of roots from Phytophthora foot-rot-affected and healthy citrus trees were analyzed by Illumina sequencing of 16S ribosomal RNA and internal transcribed spacer amplicons. Results indicate that the composition and structure of the endophytic bacterial and fungal communities were changed in roots of foot-rot-affected citrus trees. The populations of bacterial genera Asteroleplasma, Pseudomonas, and Streptomyces were decreased in foot-rot-affected trees. Also, the relative abundance of Exophiala pisciphila and Glomeraceae spp. was significantly decreased in citrus roots. Pseudomonas and Streptomyces spp. are considered beneficial bacteria and E. pisciphila and Glomeraceae spp. are dark septate endophytes and arbuscular mycorrhizal fungi, respectively, which are involved in citrus health and growth. This study provides a baseline to continue investigating the interactions between the citrus host, Phytophthora spp., and beneficial microbes.
The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora . Previous studies have demonstrated that the T3SS in E. ...amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp ⁰) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora . Expression of T3SS genes was greatly downregulated in the ppGpp ⁰ and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp ⁰ and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora . Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp -inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. IMPORTANCE The type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation of the T3SS and virulence in plant-pathogenic bacteria. Furthermore, the recovery of an spoT null mutant, which displayed very unique phenotypes, suggested that small proteins containing a single ppGpp hydrolase domain are functional.
Irrigation is important in many crop production systems. However, irrigation water can be a carrier of plant pathogens that can enter the system and spread to fields, resulting in crop damage and ...yield losses. The Lower Rio Grande Valley of South Texas is an important area for agricultural production which depends on the Rio Grande River as a source of water for irrigation. Thus, the presence of plant pathogens in the Rio Grande River could have important implications for crop productivity in the region. Cultured-based methods and molecular identification methods are used for monitoring plant pathogens in irrigation water. However, these methods are labor-intensive and just detect targeted pathogens. To overcome these limitations, in this study, the ITS2 amplicon metagenomic method was applied for evaluating the fungal diversity, composition, and presence of fungal plant pathogens in irrigation water from the Rio Grande River as it leaves the water reservoir (WR) and it arrives at an irrigation valve at a farm (FA). Results from the Shannon (WR = 4.6 ± 0.043, FA = 3.63 ± 0.13) and Simpson indices (WR = 4.6 ± 0.043, FA = 3.63 ± 0.13) showed that there are significant differences in the fungal diversity and community structure between the two locations and the PCA analysis showed a clear differentiation between both fungal communities. Several OTUs identified in both locations included potential plant pathogens from diverse genera including Cladosporium, Exserohilum, and Nigrospora, while others such as Colletotrichum and Plectosphaerella were found only in one of the two locations assessed. This work indicates that microbes, including plant pathogens, may enter or exit throughout the irrigation-water distribution system, thereby modifying the microbial community composition along the way. Understanding the dynamics of plant pathogen movement in irrigation water systems can help growers identify risk factors to develop measures to mitigate those risks. This study also shows the usefulness of the metagenomic approach for detecting and monitoring plant pathogen in irrigation water.
Huanglongbing (HLB) and Phytophthora foot and root rot are diseases that affect citrus production and profitability. The symptoms and physiological changes associated with these diseases are ...diagnosed through expensive and time-consuming field measurements. Unmanned aerial vehicles (UAVs) using red/green/blue (RGB, true color) imaging, may be an economic alternative to diagnose diseases. A methodology using a UAV with a RGB camera was developed to assess citrus health. The UAV was flown in April 2018 on a grapefruit field infected with HLB and foot rot. Ten trees were selected for each of the following disease classifications: (HLB-, foot rot–), (HLB+, foot rot–), (HLB-, foot rot+) (HLB+, foot rot+). Triangular greenness index (TGI) images were correlated with field measurements such as tree nutritional status, leaf area, SPAD (leaf greenness), foot rot disease severity and HLB. It was found that 61% of the TGI differences could be explained by Na, Fe, foot rot, Ca, and K. This study shows that diseased citrus trees can be monitored using UAVs equipped with RGB cameras, and that TGI can be used to explain subtle differences in tree health caused by multiple diseases.
Potato (
Solanum tuberosum
L.) is an important food crop worldwide. As the demand for fresh and processed potato products is increasing globally, there is a need to manage and control devastating ...diseases such as zebra chip (ZC). ZC disease causes major yield losses in many potato-growing regions and is associated with the fastidious, phloem-limited bacterium
Candidatus
Liberibacter solanacearum (
C
Lso) that is vectored by the potato-tomato psyllid (
Bactericera cockerelli
Šulc). Current management measures for ZC disease mainly focus on chemical control and integrated pest management strategies of the psyllid vector to limit the spread of
C
Lso, however, they add to the costs of potato production. Identification and deployment of
C
Lso and/or the psyllid resistant cultivars, in combination with integrated pest management, may provide a sustainable long-term strategy to control ZC. In this review, we provide a brief overview of the ZC disease, epidemiology, current management strategies, and potential new approaches to manage ZC disease in the future.
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