Publications

4485

Background/Objective. This study analyzed for the first time the asymptomatic condition induced by CLas in Citrus sinensis / C. aurantium based on initial reports in the Central Gulf region of Mexico. The objective was to analyze with a mechanistic approach the spatial-temporal concentration of CLas at the plant level in commercial orchards to demonstrate the possible implication of a prolonged incubation period in the asymptomatic expression of HLB. Materials and Methods. Two out of three trees (A1-A3) (>30 years old) of asymptomatic Valencia sweet orange trees positive for CLas, identified through official diagnostic protocols in San José Acateno, Puebla, were confined. On trees A2 and A3, 13 and 16 secondary branches were marked, with a total of 112 and 108 foliage/samples per month, respectively (total 646 samples), obtained from the distal and proximal parts of each branch. Seven monthly samplings of plant material were carried out between May and December. At the root level, three categories were sampled by diameter/function and by a combination of six distal trunk sectors (0 - 280 cm) by 4 in depth (0 x 110 cm) with a total of 111 samples. DNA extraction was performed with 2% CTAB and quantification by RT-qPCR (oligos, HLBp/HLBas). Results. In canopy, the incidence/branches in A3 was 52.2% (Ct = 22.7 – 34.9), and in A2 47.8% (Ct = 23.4 –35), with no statistical difference (p = 0.48). The inoculum load was 1.6 (±1.3) log-CLas, equivalent to 109 copies/reaction in 100 mL (range in number of copies 6.3 to 68,870.7). CLas did not differ between distal (1.6 log) and proximal (1.5 log) samples (p = 0.33). Significative temporal fluctuation was found between June and October in positive samples (37 – 87, p < 0.0001), but not in CLas concentration (1.6 – 3.5 log-CLas, p > 0.72). The distribution of CLas by branch had a temporal effect (p = 0.0001) and by sector (p = 0.07), showing high mobility and seasonality. The maximum concentration (Ymax) was associated with the middle-upper sector of the canopy, and high mobility towards the lower and upper sectors in Yinitial and Yfinal. In roots, 38.7% positivity was found. The detectability of CLas was homogeneous among root types (p < 0.0001) but variable in concentration (p = 0.04). The 47.6%, 15.4%, and 38.7% incidence was associated with 38.3, 43.4, and 9.5 average CLas copies for support, conduction, and absorption roots, respectively (p = 0.0001). Two sections with higher CLas intensity-concentration (80 – 144 copies) were associated with roots in the middle drip zone (160 – 240 cm) and medium depth strata (50 – 80 cm). Conclusion. CLas was significantly and consistently detected in plant and root tissue throughout the experimental period, but at fluctuating, non-incremental concentration, analogous between trees A2 and A3. The wide canopy-root distribution suggests a chronic systemic effect. Consequently, the incubation period does not determine the asymptomatic condition in the sweet orange/sour orange productive system investigated. Other factors, e.g. inherent to pathogen variability, could be involved. These results contribute to optimizing a surveillance-monitoring system considering seasonality, zone-branch, sector-canopy and type of tissue, with a greater probability of detection of CLas even in an eventual asymptomatic condition and generate new insights for Clas management.

Citrus Huanglongbing (HLB), a devastating disease caused by the Gram-negative bacterium ‘ Candidatus Liberibacter asiaticus’ ( C Las), poses serious threats to global citrus production and lacks effective control strategies. Previously, SDE2470 (CLIBASIA_02470) was identified as a Sec-dependent effector that contributes to C Las pathogenesis, although its underlying molecular mechanisms were not fully elucidated. In this study, SDE2470 was found to target a citrus vascular one-zinc-finger transcription factor CsVOZ2. CsVOZ2 overexpression ( CsVOZ2- OE) in transgenic citrus plants significantly suppressed C Las colonization, whereas its RNA interference (RNAi) in citrus hairy roots enhanced susceptibility to C Las. Additionally, CsVOZ2 -OE significantly increased reactive oxygen species (ROS) and abscisic acid (ABA) contents accumulation and activated related genes expression. Further investigation revealed that the E3 ligase CsBTS1 directly interacts with CsVOZ2 and promotes its degradation via the 26S proteasome pathway. CsBTS1E3 -OE in citrus hairy roots markedly enhanced C Las proliferation. Importantly, SDE2470 directly interacts with CsBTS1E3 and strengthen CsBTS1E3-CsVOZ2 interaction. Meanwhile, SDE2470 strengthened the E3 ligase activity of CsBTS1, promoting CsVOZ2 degradation. Taken together, these findings support a model in which SDE2470 hijacks CsBTS1 to destabilize CsVOZ2, thereby disrupting ROS- and ABA-dependent immunity and promoting C Las infection in citrus.

Abstract At the dawn of modern microbiology, Cohn observed abundant filamentous bacteria in drinking water wells that he named Crenothrix polyspora . Subsequent research has revealed the methanotrophic metabolism of Crenothrix bacteria and their disproportionately high activity in stratified lakes compared to unicellular strains, yet laboratory cultivation has proven elusive, leaving the ecophysiology of Crenothrix bacteria largely unknown. Here we report the isolation of two methanotroph strains of the “lacustrine Crenothrix” clade from an iron-rich wetland and reveal their highly unique cell biologies and potential ecological roles. Using physiological approaches, we demonstrate that the strains perform a microaerobic methane metabolism while growing distinct filaments having wide and directionally oriented connective structures. The strains further have broad genomic repertoires for addressing redox stress that we show are uniquely associated with lacustrine Crenothrix compared to related methanotrophs based on genome data. Aligning with laboratory observations, we identify lacustrine Crenothrix bacteria along potential redox gradients in the wetland at iron-rich snow sites, and we further detect such bacteria in diverse global ecosystems based on public metagenome searches. Together, our data strongly point to an ecophysiology of lacustrine Crenothrix bacteria that is tightly linked to redox stress, and we propose these bacteria may uniquely store or share metabolic intermediates via their filamentous lifestyle to thrive under such conditions. Our results provide a fresh view of the diversity, evolution, and ecology of aerobic methanotrophs, connecting over 150 years of microbiology research opening vast new opportunities to probe bacterial adaptations that drive global methane cycling under redox stress.

Pear decline (PD), associated with ‘Candidatus Phytoplasma pyri’, is a major disease of pear in Europe and the United States. Several psyllid species are involved in the tritrophic system of PD as vectors of phytoplasmas belonging to the 16SrX group. Four years after the first detection of PD in Sicily, an integrated approach was applied to investigate the epidemic in a major pear-growing area. Visual surveys and molecular analyses were conducted over two years in eight orchards. A total of 115 plant samples and 101 Cacopsylla spp. specimens selected from a total of 1,435 collected individuals were analysed, confirming ‘Ca. P. pyri’ in 69% of symptomatic plants and in 4.6% of C. pyri individuals. Multilocus sequence typing (MLST) revealed high genetic similarity among 16SrX isolates. Remote sensing analyses since 2018, combined with vector monitoring, confirmed the epidemic nature of PD and the persistence of a risk of further pathogen spread within the region, proving, inter alia, to be a valid method for identifying the syndrome even on a large scale.

ABSTRACT Citrus Huanglongbing (HLB), a devastating disease caused by the unculturable bacterium ‘ Candidatus Liberibacter asiaticus’ ( C Las), poses a severe threat to global citrus production. C Las secretes effectors to suppress host immune responses and facilitate its colonisation. Previously, the C Las effector SECP8 (CLIBASIA_05330) has been identified as an immune inhibitor. However, its molecular mechanisms on host immune suppression remain unclear. This study identifies the citrus transcription factor CsTCP15 as a target of SECP8. Transgenic citrus plants overexpressing CsTCP15 enhanced resistance to C Las, whereas CsTCP15 ‐RNAi interference plants became more susceptible, confirming its role as a positive immune regulator. Meanwhile, CsTCP15 was demonstrated to directly bind to cis ‐elements of salicylic acid (SA)‐responsive genes CsPR5 and CsWRKY22 , and overexpression of either gene strengthened citrus hairy roots' resistance against C Las. However, SECP8 directly interacts with CsTCP15 and inhibits its homodimerization. Concurrently, mSECP8 facilitates CsBRG3‐mediated degradation and further prevents the dimerization of CsTCP15. This two‐pronged interference eventually impairs the transcriptional activation of CsPR5 and CsWRKY22 , thereby compromising salicylic acid‐mediated immunity and promoting C Las infection. Our findings reveal a virulence strategy whereby a C Las effector manipulates a key host immune regulator to establish pathogenesis.

ABSTRACT Candidatus Loosdrechtia sp. KJ is an anaerobic ammonium-oxidizing bacterium enriched from a bioreactor operated under alkaline conditions (pH 9.2 ± 0.4). We report its 3.39 Mb draft genome, containing 3,065 predicted coding sequences, 47 tRNA genes, and a single rrn operon.